Genetically engineered P30 antigen, improved antigen cocktail, and uses thereof

ABSTRACT

The present invention relates to a genetically engineered P30 antigen and a combination or mixture of antigens (e.g., the genetically engineered P30 antigen and P35) that may be used in the detection of IgM and/or IgG antibodies to  Toxoplasma gondii.  Furthermore, the present invention also relates to methods of using this genetically engineered P30 antigen and combination of antigens, antibodies raised against this genetically engineered P30 antigen and combination of antigens, as well as kits and vaccines containing the genetically engineered P30 antigen and antigens present in the combination.

BACKGROUND OF THE INVENTION

[0001] 1. Technical Field

[0002] The present invention relates to a genetically engineered P30antigen as well as a combination or mixture of antigens which may beused in the detection of IgM and/or IgG antibodies to Toxoplasma gondii.Furthermore, the present invention also relates to methods of using thisgenetically engineered P30 antigen and combination of antigens,antibodies raised against this genetically engineered P30 antigen andcombination of antigens, as well as kits and vaccines containing thegenetically engineered P30 antigen and antigens present in thecombination.

[0003] 2. Background Information

[0004]Toxoplasma gondii is an obligate intracellular parasite which isclassified among the Coccidia. This parasite has relatively broad hostrange infecting both mammals and birds. The organism is ubiquitous innature and exists in three forms: tachyzoite, cyst, and oocyst(Remington, J. S., McLeod, R., Desmonds, G., Infectious Diseases of theFetus and Newborn Infant (J. S. Remington and J. O. Klein, Eds.), pp.140-267, Saunders, Philadelphia (1995)). Tachyzoites, found during acuteinfection, are the invasive form capable of invading all nucleatedmammalian cells. After the acute stage of infection, tissue cysts calledbradyzoites are formed within host cells and persist within the hostorganism for the life of the host. Cysts are important in transmissionof infection, especially in humans, as the ingestion of raw orundercooked meat can result in the ingestion of bradyzoites which caninfect the individual resulting in an acute infection. Oocysts representa stage of sexual reproduction which occurs only in the intestinallining of the cat family from which they are excreted in the feces.

[0005] A T. gondii infection acquired through contaminated meat or catfeces in a healthy adult is often asymptomatic. In pregnant women andimmunosuppressed patients, the clinical outcome can be very serious. Anacute infection with T. gondii acquired during pregnancy, especiallyduring the first trimester, can result in intrauterine transmission tothe unborn fetus resulting in severe fetal and neonatal complications,including mental retardation and fetal death. Recrudescence of aprevious T. gondii infection or an acute infection in animmunosuppressed individual can be pathogenic. Toxoplasmic encephalitisis a major cause of morbidity and mortality in AIDS patients. Toxoplasmainfection has also been shown to be a significant cause ofchorioretinitis in children and adults.

[0006] Diagnosis of infection with T. gondii may be established by theisolation of T. gondii from blood or body fluids, demonstration of thepresence of the organism in the placenta or tissues of the fetus,demonstration of the presence of antigen by detection of specificnucleic acid sequences (e.g., DNA probes), or detection of T. gondiispecific immunoglobulins synthesized by the host in response toinfection using serologic tests.

[0007] The detection of T. gondii specific antibodies and determinationof antibody titer are important tools used in the diagnosis oftoxoplasmosis. The most widely used serologic tests for the diagnosis oftoxoplasmosis are the Sabin-Feldman dye test (Sabin, A. B. and Feldman,H. A. (1948) Science 108, 660-663), the indirect hemagglutination (IHA)test (Jacobs, L. and Lunde, M. (1957) J. Parasitol. 43, 308-314), theIFA test (Walton, B. C. et al. (1966) Am. J. Trop. Med. Hyg. 15,149-152), the agglutination test (Fondation Mérieux, Sérologie deI'Infection Toxoplasmique en Particulier à Son Début: Méthodes etInterprétation des Résultants, Lyon, 182 pp. (1975)) and the ELISA(Naot, Y. and Remington, J. S. (1980) J. Infect. Dis. 142, 757-766). TheELISA test is one the easiest tests to perform, and many automatedserologic tests for the detection of Toxoplasma specific IgM and IgG arecommercially available.

[0008] The current tests for the detection of IgM and IgG antibodies ininfected individuals can vary widely in their ability to detect serumantibody. Hence, there is significant inter-assay variation seen amongthe commercially available kits. The differences observed between thedifferent commercial kits are caused primarily by the preparation of theantigen used for the serologic test. Most kits use either whole orsonicated tachyzoites grown in tissue culture or in mice which contain ahigh proportion of extra-parasitic material, for example, mammaliancells, tissue culture components, etc. Due to the lack of a purified,standardized antigen or standard method for preparing the tachyzoiteantigen, it is not surprising that inter-assay variability existsresulting in different assays having different performancecharacteristics in terms of assay sensitivity and specificity.

[0009] Given the limitations of serologic tests employing the tachyzoiteantigen, as described above, as well as the persistent problemsregarding determination of onset of infection, purified recombinantantigens obtained by molecular biology are an attractive alternative inthat they can be purified and standardized. In the literature, a numberof Toxo genes have been cloned and expressed in a suitable host toproduce immunoreactive, recombinant Toxo antigens. For example, the ToxoP22 (SAG2), P24 (GRA1), P25, P28 (GRA2), P29 (GRA7), P30 (SAG1), P35,P41 (GRA4), P54 (ROP2), P66 (ROP1), and the Toxo P68 antigens have beendescribed (Prince et al. (1990) Mol. Biochem. Parasitol 43, 97-106;Cesbron-Delauw et al. (1989) Proc. Nat. Acad. Sci. 86, 7537-7541;Johnson et al. (1991) Gene 99, 127-132; Prince et al. (1989) Mol.Biochem. Parasitol. 34, 3-13; Bonhomme et al. (1998) J. Histochem.Cytochem. 46, 1411-1421; Burg et al. (1988) J. Immunol. 141, 3584-3591;Knapp et al. (1989) EPA 431541A2; Mevelec et al. (1992) Mol. Biochem.Parasitol. 56, 227-238; Saavedra et al. (1991) J. Immunol. 147,1975-1982); EPA 751 147).

[0010] It is plausible that no single Toxo antigen can replace thetachyzoite in an initial screening immunoassay for the detection ofToxo-specific immunoglobulins. This may be due to several reasons.First, the antibodies produced during infection vary with the stage ofinfection, i.e., the antibodies produced by an infected individual varyover time reacting with different epitopes. Secondly, the epitopespresent in a recombinant antigen may be different or less reactive thannative antigen prepared from the tachyzoite depending on the host usedfor expression and the purification scheme employed. Thirdly, differentrecombinant antigens may be needed to detect the different classes ofimmunoglobulins produced in response to an infection, e.g., IgM, IgG,IgA and IgE.

[0011] In order to overcome the limitations of the tachyzoite antigen interms of assay specificity and sensitivity, a search was done for Toxoantigens which could be used in combination in order to configure newassays for the detection of Toxo-specific immunoglobulins. Maine et al.(in U.S. Pat. No. 6,329,157 B1) disclose recombinant Toxo antigencocktails for the detection of Toxo-specific IgG and IgM. It wasdetermined that the above mentioned Toxo antigen cocktails could beimproved and enhanced by expression of Toxo P30 in E. coli as a solubleprotein with genetically engineered modifications. This geneticallyengineered P30 antigen and improved antigen cocktail will be describedin further detail below.

SUMMARY OF THE INVENTION

[0012] The present invention includes a genetically engineeredToxoplasma gondii P30 antigen as well as a composition comprising bothToxoplasma gondii genetically engineered P30 antigen and P35 antigen.This genetically engineered antigen and composition may be used asdiagnostic reagents, and the genetically engineered antigen and theantigens within this composition may be produced either recombinantly orsynthetically.

[0013] In particular, the present invention includes an isolatednucleotide sequence or fragment thereof comprising or complementary to anucleotide sequence having at least 70% nucleotide sequence identity toa nucleotide sequence selected from the group consisting of SEQ IDNO:22, SEQ ID NO:27 and SEQ ID NO:63. The present invention alsoincludes an isolated nucleotide sequence or fragment thereof encoding apolypeptide, wherein the polypeptide has at least 70% amino acidsequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64. The presentinvention also includes a purified polypeptide encoded by any of thenucleotide sequences presented above.

[0014] Additionally, the present invention includes a purifiedpolypeptide or fragment thereof having at least 70% amino acid sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64. Also, the present inventionincludes a purified polypeptide or fragment thereof comprising an aminoacid sequence having 1-6 additional amino acids at the C-terminus of SEQID NO:28. The invention also includes a purified polypeptide or fragmentthereof comprising an amino acid sequence as in SEQ ID NO:23 in whichany one or more of the five C-terminal amino acids have been changedfrom cysteine to alanine. Further, the present invention also includes apolyclonal or monoclonal antibody directed against these purifiedpolypeptides.

[0015] The present invention also includes a composition comprising apolypeptide, wherein the polypeptide comprises an amino acid sequenceselected from the group consisting of SEQ ID NO:23, SEQ ID NO:28 and SEQID NO:64. This composition may be used as a diagnostic reagent, and thepolypeptide of the composition may be produced by recombinant orsynthetic means.

[0016] Additionally, the present invention includes a method fordetecting the presence of IgM antibodies to Toxoplasma gondii in a testsample comprising the steps of: a) contacting the test sample suspectedof containing the IgM antibodies with a composition comprising apolypeptide, wherein the polypeptide comprises an amino acid sequencehaving at least 70% amino acid identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:23, SEQ ID NO:28 and SEQID NO:64; and b) detecting the presence of polypeptide/IgM antibodycomplexes, wherein presence of the complexes indicates presence of theIgM antibodies in the test sample.

[0017] Furthermore, the present invention also includes a method fordetecting the presence of IgM antibodies to Toxoplasma gondii in a testsample comprising the steps of: a) contacting the test sample suspectedof containing the IgM antibodies with a composition comprising apolypeptide, wherein the polypeptide comprises an amino acid sequencehaving at least 70% amino acid identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:23, SEQ ID NO:28 and SEQID NO:64, for a time and under conditions sufficient for the formationof IgM antibody/antigen complexes; b) adding a conjugate to theresulting IgM antibody/antigen complexes for a time and under conditionssufficient to allow said conjugate to bind to the bound antibody,wherein the conjugate comprises an antibody attached to asignal-generating compound capable of generating a detectable signal;and c) detecting presence of IgM antibodies which may be present in thetest sample by detecting presence of a signal generated by thesignal-generating compound.

[0018] Moreover, the present invention encompasses a method fordetecting the presence of IgG antibodies to Toxoplasma gondii in a testsample comprising the steps of: a) contacting the test sample suspectedof containing the IgG antibodies with a composition comprising: 1) apolypeptide, wherein the polypeptide comprises an amino acid sequencehaving at least 70% amino acid identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:23, SEQ ID NO:28 and SEQID NO:64 and 2) P35; and b) detecting presence of antigen/IgG antibodycomplexes, presence of the complexes indicating presence of said IgGantibodies in the test sample.

[0019] The invention also encompasses a method for detecting thepresence of IgG antibodies to Toxoplasma gondii in a test samplecomprising the steps of: a) contacting the test sample suspected ofcontaining the IgG antibodies with a composition comprising: 1) apolypeptide, wherein the polypeptide comprises an amino acid sequencehaving at least 70% amino acid identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:23, SEQ ID NO:28 and SEQID NO:64 and 2) P35, for a time and under conditions sufficient forformation of IgG antibody/antigen complexes; b) adding a conjugate toresulting IgG antibody/antigen complexes for a time and under conditionssufficient to allow the conjugate to bind to bound antibody, wherein theconjugate comprises an antibody attached to a signal-generating compoundcapable of generating a detectable signal; and c) detecting IgGantibodies which may be present in the test sample by detecting presenceof a signal generated by said signal-generating compound.

[0020] Furthermore, the present invention includes a method fordetecting the presence of IgM antibodies to Toxoplasma gondii in a testsample comprising the steps of: a) contacting the test sample suspectedof containing the IgM antibodies with anti-antibody specific for the IgMantibodies for a time and under conditions sufficient to allow forformation of anti-antibody/IgM antibody complexes; b) adding a conjugateto resulting anti-antibody/IgM antibody complexes for a time and underconditions sufficient to allow the conjugate to bind to bound antibody,wherein the conjugate comprises a polypeptide, wherein the polypeptidecomprises an amino acid sequence having at least 70% amino acid sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64, attached to a signalgenerating compound capable of generating a detectable signal; and c)detecting IgM antibodies which may be present in the test sample bydetecting presence of a signal generated by the signal-generatingcompound.

[0021] Further, the present invention includes a method for detectingthe presence of IgG antibodies to Toxoplasma gondii in a test samplecomprising the steps of: a) contacting the test sample suspected ofcontaining the IgG antibodies with anti-antibody specific for the IgGantibodies for a time and under conditions sufficient to allow forformation of anti-antibody/IgG antibody complexes; b) adding a conjugateto resulting anti-antibody/IgG antibody complexes for a time and underconditions sufficient to allow the conjugate to bind to bound antibody,wherein the conjugate comprises: 1) a polypeptide, wherein thepolypeptide comprises an amino acid sequence having at least 70% aminoacid sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64, and 2) P35,each attached to a signal-generating compound capable of generating adetectable signal; and c) detecting IgG antibodies which may be presentin the test sample by detecting the presence of a signal generated byeach of the signal-generating compounds.

[0022] The present invention also encompasses a vaccine comprising: a)at least one polypeptide selected from the group consisting of: 1) apolypeptide, wherein the polypeptide comprises amino acid sequencehaving at least 70% amino acid sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO:23, SEQ IDNO:28 and SEQ ID NO:64 and 2) P35, and b) a pharmaceutically acceptableadjuvant.

[0023] Also, the invention includes a kit for determining the presenceof IgM antibodies to Toxoplasma gondii in a test sample comprising acomposition comprising a polypeptide, wherein the polypeptide comprisesan amino acid sequence having at least 70% amino acid sequence identityto an amino acid sequence selected from the group consisting of SEQ IDNO:23, SEQ ID NO:28 and SEQ ID NO:64; and a conjugate comprising anantibody attached to a signal-generating compound capable of generatinga detectable signal.

[0024] Also, the present invention includes a kit for determining thepresence of IgG antibodies to Toxoplasma gondii in a test samplecomprising: a composition comprising 1) a polypeptide, wherein thepolypeptide comprises an amino acid sequence having at least 70% aminoacid sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64 and 2) P35;and a conjugate comprising an antibody attached to a signal-generatingcompound capable of generating a detectable signal.

[0025] Additionally, the present invention encompasses a kit fordetermining the presence of IgM antibodies to Toxoplasma gondii in atest sample comprising:

[0026] a) an anti-antibody specific for IgM antibody; and

[0027] b) a composition comprising a polypeptide, wherein thepolypeptide comprises an amino acid sequence having at least 70% aminoacid sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64.

[0028] Furthermore, the present invention includes a kit for determiningthe presence of IgM antibodies to Toxoplasma gondii in a test samplecomprising:

[0029] a) an anti-antibody specific for IgM antibody;

[0030] b) a conjugate comprising: 1) a composition comprising apolypeptide, wherein the polypeptide comprises an amino acid sequencehaving at least 70% amino acid sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO:23, SEQ IDNO:28 and SEQ ID NO:64, attached to 2) a signal-generating compoundcapable of generating a detectable signal.

[0031] Also, the invention includes a kit for determining the presenceof IgG antibodies to Toxoplasma gondii in a test sample comprising:

[0032] a) an anti-antibody specific for IgG antibody; and

[0033] b) a composition comprising: 1) a polypeptide, wherein saidpolypeptide comprises an amino acid sequence having at least 70% aminoacid sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64 and 2) P35.

[0034] Another kit of the present invention includes a kit fordetermining the presence of IgG antibodies to Toxoplasma gondii in atest sample comprising:

[0035] a) an anti-antibody specific for IgG antibody;

[0036] b) a conjugate comprising: 1) a polypeptide, wherein thepolypeptide comprises an amino acid sequence having at least 70% aminoacid sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64, and 2) P35,each attached to a signal-generating compound capable of generating adetectable signal.

[0037] Further, the present invention includes a method for detectingthe presence of IgM antibodies to Toxoplasma gondii in a test samplecomprising the steps of: (a) contacting said test sample suspected ofcontaining IgM antibodies with anti-antibody specific for the IgMantibodies for a time and under conditions sufficient to allow forformation of anti-antibody IgM complexes; (b) adding antigen toresulting anti-antibody/IgM complexes for a time and under conditionssufficient to allow the antigen to bind to bound IgM antibody, theantigen comprising a polypeptide, wherein the polypeptide comprises anamino acid sequence having at least 70% amino acid sequence identity toan amino acid sequence selected from the group consisting of SEQ IDNO:23, SEQ ID NO:28 and SEQ ID NO:64; and (c) adding a conjugate toresulting anti-antibody/IgM/antigen complexes, the conjugate comprisinga composition comprising monoclonal or polyclonal antibody attached to asignal-generating compound capable of generating a detectable signal;and (d) detecting IgM antibodies which may be present in the test sampleby detecting a signal generated by the signal-generating compound.

[0038] Additionally, the invention includes a method for detecting thepresence of IgG antibodies to Toxoplasma gondii in a test samplecomprising the steps of: (a) contacting the test sample suspected ofcontaining IgG antibodies with anti-antibody specific for the IgGantibodies for a time and under conditions sufficient to allow forformation of anti-antibody IgG complexes; (b) adding antigen toresulting anti-antibody/IgG complexes for a time and under conditionssufficient to allow the antigen to bind to bound IgG antibody, theantigen comprising a mixture of 1) a polypeptide, wherein thepolypeptide comprises an amino acid sequence having at least 70% aminoacid sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64, and 2) P35;(c) adding a conjugate to resulting anti-antibody/IgG/antigen complexes,the conjugate comprising a composition comprising a monoclonal orpolyclonal antibody attached to a signal-generating compound capable ofgenerating a detectable signal; and (d) detecting IgG antibodies whichmay be present in the test sample by detecting a signal generated by thesignal-generating compound.

[0039] The present invention also includes a method for detecting thepresence of IgM and IgG antibodies to Toxoplasma gondii in a test samplecomprising the steps of: a) contacting the test sample suspected ofcontaining said IgM and IgG antibodies with a composition comprising 1)a polypeptide, wherein said polypeptide comprises an amino acid sequencehaving at least 70% amino acid sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO:23, SEQ IDNO:28 and SEQ ID NO:64, and 2) P35, for a time and under conditionssufficient for the formation of IgM and IgG antibody/antigen complexes;b) adding a conjugate to the resulting IgM antibody/antigen complexesand IgG antibody/antigen complexes for a time and under conditionssufficient to allow the conjugate to bind to the bound IgM and IgGantibody, wherein the conjugate comprises an antibody attached to asignal-generating compound capable of generating a detectable signal;and c) detecting the presence of IgM and IgG antibodies which may bepresent in said test sample by detecting a signal generated by thesignal-generating compound.

[0040] The invention also encompasses a method for detecting thepresence of IgM and IgG antibodies to Toxoplasma gondii in a test samplecomprising the steps of: a) contacting the test sample suspected ofcontaining the IgM and IgG antibodies with anti-antibody specific forthe IgM antibodies and the IgG antibodies for a time and underconditions sufficient to allow for formation of anti-antibody/IgMantibody complexes and anti-antibody/IgG antibody complexes; b) adding aconjugate to resulting anti-antibody/IgM antibody complexes andresulting anti-antibody/IgG antibody complexes for a time and underconditions sufficient to allow the conjugate to bind to bound antibody,wherein the conjugate comprises a composition comprising: 1) apolypeptide, wherein the polypeptide comprises an amino acid sequencehaving at least 70% amino acid sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO:23, SEQ IDNO:28 and SEQ ID NO:64, and 2) P35, each attached to a signal-generatingcompound capable of generating a detectable signal; and c) detecting IgMand IgG antibodies which may be present in the test sample by detectinga signal generated by the signal-generating compound.

[0041] Moreover, the present invention also encompasses a method fordetecting the presence of IgM and IgG antibodies to Toxoplasma gondii ina test sample comprising the steps of: (a) contacting the test samplesuspected of containing IgM and IgG antibodies with anti-antibodyspecific for the IgM antibodies and with anti-antibody specific for theIgG antibodies for a time and under conditions sufficient to allow forformation of anti-antibody/IgM complexes and anti-antibody/IgGcomplexes; (b) adding antigen to resulting anti-antibody/IgM complexesand resulting anti-antibody/IgG complexes for a time and underconditions sufficient to allow said antigen to bind to bound IgM and IgGantibody, the antigen comprising a mixture of: 1) a polypeptide, whereinsaid polypeptide comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64 and 2)P35; and (c) adding a conjugate to resulting anti-antibody/IgM/antigencomplexes and anti-antibody/IgG/antigen complexes, the conjugatecomprising a composition comprising monoclonal or polyclonal antibodyattached to a signal-generating compound capable of generating adetectable signal; and (d) detecting IgM and IgG antibodies which may bepresent in the test sample by detecting a signal generated by thesignal-generating compound.

[0042] The present invention also includes a method of producingmonoclonal antibodies comprising the steps of injecting a non-humanmammal with a polypeptide, wherein the polypeptide comprises an aminoacid sequence having at least 70% amino acid sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NO:23,SEQ ID NO:28 and SEQ ID NO:64; fusing spleen cells of the non-humanmammal with myeloma cells in order to generate hybridomas; and culturingthe hybridomas for a time and under conditions sufficient for thehybridomas to produce the monoclonal antibodies.

[0043] Moreover, the present invention encompasses the plasmidpMBP-c2X-ToxoP30del3C(52-300aa), the plasmidpMBP-c2X-ToxoP30del4C(52-294aa), as well as the plasmidpMBP-c2X-ToxoP30MIX1.

[0044] The invention also includes an isolated nucleotide sequencecomprising or complementary to the nucleotide sequence of SEQ ID NO:20as well as a purified polypeptide comprising the amino acid sequence ofSEQ ID NO:21.

[0045] Furthermore, the present invention includes an isolatednucleotide sequence comprising or complementary to the nucleotidesequence of SEQ ID NO:25 as well as a purified polypeptide comprisingthe amino acid sequence of SEQ ID NO:26.

[0046] Additionally, the invention includes an isolated nucleotidesequence comprising or complementary to the nucleotide sequence of SEQID NO:61 as well as a purified polypeptide comprising the amino acidsequence of SEQ ID NO:62.

[0047] The present invention also includes portions or fragments ofToxoP30del3C(52-300aa), ToxoP30del4C(52-294aa), or ToxoP30MIX1, whichhave the same antigenic properties as the region ofToxoP30del3C(52-300aa) which consists of amino acids 1-249, as theregion of ToxoP30del4C(52-294aa) which consists of amino acids 1-243,and the region of ToxoP30MIX1 which consists of amino acids 1-249,respectively.

[0048] All U.S. patents and publications referred to herein are herebyincorporated in their entirety by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049]FIG. 1 is a schematic of the construction of plasmidpMBP-c2X-ToxoP30(52-336aa). (The amino acid range in parentheses, notedhere and throughout the application, refers to the amino acid sequence(e.g., present in the plasmid, protein, etc.) which has been derivedfrom the native P30 antigen.)

[0050]FIG. 2 represents the DNA sequence [SEQ ID NO:3] of nucleotides1-7478 encoding the amino acid sequence [SEQ ID NO:4] of theMBP-ToxoP30(52-336aa) fusion protein of plasmidpMBP-c2X-ToxoP30(52-336aa).

[0051]FIG. 3 represents the DNA sequence [SEQ ID NO:5] of nucleotides1-850 of the ToxoP30(52-336aa) gene and the corresponding encoded aminoacid sequence [SEQ ID NO:6] of the ToxoP30(52-336aa) protein.

[0052]FIG. 4 is a schematic of the construction of plasmidpMBP-p2X-ToxoP30(52-336aa).

[0053]FIG. 5 represents the DNA sequence [SEQ ID NO:7] of nucleotides1-7553 and the corresponding encoded amino acid sequence [SEQ ID NO:8]of the MBP-ToxoP30(52-336aa) fusion protein of plasmidpMBP-p2X-ToxoP30(52-336aa).

[0054]FIG. 6 is a schematic of the construction of plasmidpMBP-c2X-ToxoP30del1(52-324aa).

[0055]FIG. 7 is a schematic of the construction of plasmidpMBP-c2X-ToxoP30del1C(52-324aa).

[0056]FIG. 8 represents the DNA sequence [SEQ ID NO:10] of nucleotides1-7442 and the corresponding encoded amino acid sequence [SEQ ID NO:11]of the MBP-ToxoP30del1C(52-324aa) fusion protein of plasmidpMBP-c2X-ToxoP30del1C(52-324aa).

[0057]FIG. 9 represents the DNA sequence [SEQ ID NO:12] of nucleotides1-819 of the ToxoP30del1C(52-324) gene and the corresponding encodedamino acid sequence [SEQ ID NO:13] of the ToxoP30del1C(52-324aa)protein.

[0058]FIG. 10 is a schematic of the construction of plasmidpMBP-c2X-ToxoP30del2(52-311aa).

[0059]FIG. 11 represents the DNA sequence [SEQ ID NO:15] of nucleotides1-7403 and the corresponding encoded amino acid sequence [SEQ ID NO:16]of the MBP-ToxoP30del2(52-311aa) fusion protein of plasmidpMBP-c2X-ToxoP30del2(52-311aa).

[0060]FIG. 12 represents the DNA sequence [SEQ ID NO:17] of nucleotides1-780 of the ToxoP30del2(52-311aa) gene and the corresponding encodedamino acid sequence [SEQ ID NO:18] of the ToxoP30del2(52-311aa) protein.

[0061]FIG. 13 is a schematic of the construction of plasmidpMBP-c2X-ToxoP30del3(52-300aa).

[0062]FIG. 14 is a schematic of the construction of plasmidpMBP-c2X-ToxoP30del3C(52-300aa).

[0063]FIG. 15 represents the DNA sequence [SEQ ID NO:20] of nucleotides1-7370 and the corresponding encoded amino acid sequence [SEQ ID NO:21]of the MBP-ToxoP30del3C(52-300aa) fusion protein of plasmidpMBP-c2X-ToxoP30del3C(52-300aa).

[0064]FIG. 16 represents the DNA sequence [SEQ ID NO:22] of nucleotides1-747 of the ToxoP30del3(52-300aa) and the corresponding encoded aminoacid sequence [SEQ ID NO:23] of the ToxoP30del3C(52-300aa) protein.

[0065]FIG. 17 is a schematic of the construction of plasmidpMBP-c2X-ToxoP30del4(52-294aa).

[0066]FIG. 18 is a schematic of the construction of plasmidpMBP-c2X-ToxoP30del4C(52-294aa).

[0067]FIG. 19 represents the DNA sequence [SEQ ID NO:25] of nucleotides1-7352 and the corresponding encoded amino acid sequence [SEQ ID NO:26]of the MBP-ToxoP30del4C(52-294aa) fusion protein of plasmidpMBP-c2X-ToxoP30del4C(52-294aa).

[0068]FIG. 20 represents the DNA sequence [SEQ ID NO:27] of nucleotides1-729 of the ToxoP30del4C(52-294aa) gene and the corresponding encodedamino acid sequence [SEQ ID NO:28] of the ToxoP30del4C(52-294aa)protein.

[0069]FIG. 21 is a schematic of the construction of plasmidpMBP-c2X-ToxoP30del4del8(83-294aa).

[0070]FIG. 22 represents the DNA sequence [SEQ ID NO:30] of nucleotides1-7259 and the corresponding encoded amino acid sequence [SEQ ID NO:31]of the MBP-ToxoP30del4del8(83-294aa) fusion protein of plasmidpMBP-c2X-ToxoP30del4del8(83-294aa).

[0071]FIG. 23 represents the DNA sequence [SEQ ID NO:32] of nucleotides1-636 of the ToxoP30del4del8(83-294aa) gene and the corresponding aminoacid sequence [SEQ ID NO:33] of the ToxoP30del4del8(83-294aa) protein.

[0072]FIG. 24 is a schematic of the construction of plasmidpMBP-c2X-ToxoP30del10(52-284aa).

[0073]FIG. 25 represents the DNA sequence [SEQ ID NO:35] of nucleotides1-7322 and the corresponding encoded amino acid sequence [SEQ ID NO:36]of the MBP-ToxoP30del10(52-284aa) fusion protein of plasmidpMBP-c2X-ToxoP30del10(52-284aa).

[0074]FIG. 26 represents the DNA sequence [SEQ ID NO:37] of nucleotides1-699 of the ToxoP30del10(52-284aa) gene and the corresponding encodedamino acid sequence [SEQ ID NO:38] of the ToxoP30del10(52-284aa)protein.

[0075]FIG. 27 is a schematic of the construction of plasmidpMBP-c2X-ToxoP30del11(52-214aa).

[0076]FIG. 28 represents the DNA sequence [SEQ ID NO:40] of nucleotides1-7112 and the corresponding encoded amino acid sequence [SEQ ID NO:41]of the MBP-ToxoP30del11(52-214aa) fusion protein of plasmidpMBP-c2X-ToxoP30del11(52-214aa).

[0077]FIG. 29 represents the DNA sequence [SEQ ID NO:42] of nucleotides1-489 of the ToxoP30del11(52-214aa) gene and the corresponding encodedamino acid sequence [SEQ ID NO:43] of the ToxoP30del11(52-214aa)protein.

[0078]FIG. 30 is a schematic of the construction of plasmidspMBP-c2X-ToxoP30MIX1, pMBP-c2X-ToxoP30MIX3, and pMBP-c2X-ToxoP30MIX5.

[0079]FIG. 31 represents the DNA sequence [SEQ ID NO:61] of nucleotides1-7370 and the corresponding encoded amino acid sequence [SEQ ID NO:62]of the MBP-ToxoP30MIX1 fusion protein of plasmid pMBP-c2X-ToxoP30MIX1.

[0080]FIG. 32 represents the DNA sequence [SEQ ID NO:63] of nucleotides1-747 of the ToxoP30MIX1 gene and the corresponding encoded amino acidsequence [SEQ ID NO:64] of the ToxoP30MIX1 protein.

[0081]FIG. 33 represents the DNA sequence [SEQ ID NO:66] of nucleotides1-7370 and the corresponding encoded amino acid sequence [SEQ ID NO:67]of the MBP-ToxoP30MIX3 fusion protein of plasmid pMBP-c2X-ToxoP30MIX3.

[0082]FIG. 34 represents the DNA sequence [SEQ ID NO:68] of nucleotides1-747 of the ToxoP30MIX3 gene and the corresponding amino acid sequence[SEQ ID NO:69] of the ToxoP30MIX3 protein.

[0083]FIG. 35 represents the DNA sequence [SEQ ID NO:71] of nucleotides1-7370 and the corresponding encoded amino acid sequence [SEQ ID NO:72]of the MBP-ToxoP30MIX5 fusion protein of plasmid pMBP-c2X-ToxoP30MIX5.

[0084]FIG. 36 represents the DNA sequence [SEQ ID NO:73] of nucleotides1-747 of the ToxoP30MIX5 gene and the corresponding encoded amino acidsequence [SEQ ID NO:74] of the ToxoP30MIX5 protein.

DETAILED DESCRIPTION OF THE INVENTION

[0085] The difficulties of known assays for the detection of IgG and IgMantibodies to T. gondii have been described, in detail, above. Thus,there was a need to discover immunoassays that could accurately detectthe presence of such antibodies in positive serum or plasma, therebyeliminating the problem of false negative or false positive tests. Thepresent invention provides such needed immunoassays and, in particular,an antigen and combinations of antigens which accurately detect thepresence of IgG and/or IgM antibodies in human sera.

[0086] In particular, the present invention includes geneticallyengineered versions of the P30 antigen referred to herein as“ToxoP30del3C(52-300aa)” and “ToxoP30del4C(52-294aa)”, which containsmall and precise deletions at the C-terminus of each protein thatmaximize the anti-Toxo IgG and IgM immunoreactivity of the P30 antigenin an immunoassay. The present invention also includes a geneticallyengineered version of the P30 antigen referred to herein as“ToxoP30MIX1”, which contains the same deletion at the C-terminus asToxoP30del3C(52-300aa) as well as five C-terminal cysteine residueschanged to alanine. The invention also includes a polypeptide comprisingthe amino acid sequence of ToxoP30del3C(52-300aa) in which any one ormore of the last five cysteines at the C-terminus have been changed toalanine.

[0087] The nucleotide sequence of the gene encoding the ToxoP30del3Cantigen is shown in FIG. 16 and is represented by SEQ ID NO:22. Theamino acid sequence of this antigen is also shown in FIG. 16 and isrepresented by SEQ ID NO:23. The nucleotide sequence of the geneencoding the ToxoP30del4C antigen is shown in FIG. 20 and is representedby SEQ ID NO:27. The amino acid sequence of this antigen is also shownin FIG. 20 and is represented by SEQ ID NO: 28. The nucleotide sequenceof the gene encoding the ToxoP30MIX1 antigen is shown in FIG. 32 and isrepresented by SEQ ID NO:63. The amino acid sequence of this antigen isalso shown in FIG. 32 and is represented by SEQ ID NO:64.

[0088] It should be noted that the present invention also encompassesnucleotide sequences comprising or complementary to a nucleotidesequence having at least about 70% nucleotide sequence identity,preferably at least about 80% nucleotide sequence identity, and morepreferably at least about 90% nucleotide sequence identity to thenucleotide sequence of SEQ ID NO:22, SEQ ID NO:27 or SEQ ID NO:63. (Allintegers within the ranges noted above (i.e., between 70 and 100) arealso considered to fall within the scope of the present invention.) Thesequence having the above-described percent identity or complementarysequences may be derived from species or sources other than from whichthe isolated, original sequences were derived.

[0089] Also, it should be noted that the present invention encompasses apolypeptide sequence comprising an amino acid sequence having at leastabout 70% amino acid sequence identity, preferably at least about 80%amino acid sequence identity, and more preferably at least about 90%amino acid sequence identity to the amino acid sequence of SEQ ID NO:23,SEQ ID NO:28 or SEQ ID NO:64. (All integers within the ranges notedabove (i.e, between 70 and 100) are also considered to fall within thescope of the present invention.)

[0090] For purposes of the present invention, “complementarity” isdefined as the degree of relatedness between two DNA segments. It isdetermined by measuring the ability of the sense strand of one DNAsegment to hybridize with the antisense strand of the other DNA segment,under appropriate conditions, to form a double helix. In the doublehelix, wherever adenine appears in one strand, thymine appears in theother strand. Similarly, wherever guanine is found in one strand,cytosine is found in the other. The greater the relatedness between thenucleotide sequences of two DNA segments, the greater the ability toform hybrid duplexes between the strands of two DNA segments.

[0091] The term “identity” refers to the relatedness of two sequences ona nucleotide-by-nucleotide basis over a particular comparison window orsegment. Thus, identity is defined as the degree of sameness,correspondence or equivalence between the same strands (either sense orantisense) of two DNA segments (or two amino acid sequences).“Percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over a particular region, determining thenumber of positions at which the identical base or amino acid occurs inboth sequences in order to yield the number of matched positions,dividing the number of such positions by the total number of positionsin the segment being compared and multiplying the result by 100. Optimalalignment of sequences may be conducted by the algorithm of Smith &Waterman, Appl. Math. 2:482 (1981), by the algorithm of Needleman &Wunsch, J. Mol. Biol. 48:443 (1970), by the method of Pearson & Lipman,Proc. Natl. Acad. Sci. (USA) 85:2444 (1988) and by computer programswhich implement the relevant algorithms (e.g., Clustal Macaw Pileup(http://cmgm.stanford.edu/biochem218/11Multiple.pdf; Higgins et al.,CABIOS. 5L151-153 (1989)), FASTDB (Intelligenetics), BLAST (NationalCenter for Biomedical Information; Altschul et al., Nucleic AcidsResearch 25:3389-3402 (1997)), PILEUP (Genetics Computer Group, Madison,Wis.) or GAP, BESTFIT, FASTA and TFASTA (Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, Madison, Wis.). (See U.S.Pat. No. 5,912,120.)

[0092] “Similarity” between two amino acid sequences is defined as thepresence of a series of identical as well as conserved amino acidresidues in both sequences. The higher the degree of similarity betweentwo amino acid sequences, the higher the correspondence, sameness orequivalence of the two sequences. (“Identity between two amino acidsequences is defined as the presence of a series of exactly alike orinvariant amino acid residues in both sequences.) The definitions of“complementarity”, “identity” and “similarity” are well known to thoseof ordinary skill in the art.

[0093] “Encoded by” refers to a nucleic acid sequence which codes for apolypeptide sequence, wherein the polypeptide sequence or a portionthereof contains an amino acid sequence of at least 3 amino acids, morepreferably at least 8 amino acids, and even more preferably at least 15amino acids from a polypeptide encoded by the nucleic acid sequence.

[0094] The present invention also encompasses an isolated nucleotidesequence which is hybridizable, under moderately stringent conditions,to a nucleic acid having a nucleotide sequence comprising orcomplementary to the nucleotide sequences described above. A nucleicacid molecule is “hybridizable” to another nucleic acid molecule when asingle-stranded form of the nucleic acid molecule can anneal to theother nucleic acid molecule under the appropriate conditions oftemperature and ionic strength (see Sambrook et al., “Molecular Cloning:A Laboratory Manual, Second Edition (1989), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.)). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. “Hybridization” requires that two nucleic acids containcomplementary sequences. However, depending on the stringency of thehybridization, mismatches between bases may occur. The appropriatestringency for hybridizing nucleic acids depends on the length of thenucleic acids and the degree of complementation. Such variables are wellknown in the art. More specifically, the greater the degree ofsimilarity or homology between two nucleotide sequences, the greater thevalue of Tm for hybrids of nucleic acids having those sequences. Forhybrids of greater than 100 nucleotides in length, equations forcalculating Tm have been derived (see Sambrook et al., supra). Forhybridization with shorter nucleic acids, the position of mismatchesbecomes more important, and the length of the oligonucleotide determinesits specificity (see Sambrook et al., supra).

[0095] As used herein, an “isolated nucleic acid fragment or sequence”is a polymer of RNA or DNA that is single- or double-stranded,optionally containing synthetic, non-natural or altered nucleotidebases. An isolated nucleic acid fragment in the form of a polymer of DNAmay be comprised of one or more segments of cDNA, genomic DNA orsynthetic DNA. (A “fragment” of a specified polynucleotide refers to apolynucleotide sequence which comprises a contiguous sequence ofapproximately at least about 6 nucleotides, preferably at least about 8nucleotides, more preferably at least about 10 nucleotides, and evenmore preferably at least about 15 nucleotides, and most preferably atleast about 25 nucleotides identical or complementary to a region of thespecified nucleotide sequence. (See U.S. Pat. No. 6,183,952 B1.) Incontrast, a “fragment” of a specified polypeptide refers to an aminoacid sequence which comprises at least about 5 amino acids, morepreferably at least about 10 amino acids, and even more preferably atleast 15 amino acids derived from the specified polypeptide.)Nucleotides (usually found in their 5′-monophosphate form) are referredto by their single letter designation as follows: “A” for adenylate ordeoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate ordeoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate,“T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines(C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N”for any nucleotide.

[0096] The terms “fragment or subfragment that is functionallyequivalent” and “functionally equivalent fragment or subfragment” areused interchangeably herein. These terms refer to a portion orsubsequence of an isolated nucleic acid fragment in which the ability toalter gene expression or produce a certain phenotype is retained whetheror not the fragment or subfragment encodes an active enzyme. A fragmentor subfragment that is functionally equivalent to the originalpolypeptide sequence from which it is derived refers to a sequence whichhas the same properties (e.g., binding, antigenic, etc.) as the originalpolypeptide.

[0097] The terms “homology”, “homologous”, “substantially similar” and“corresponding substantially” are used interchangeably herein. Theyrefer to nucleic acid fragments wherein changes in one or morenucleotide bases does not affect the ability of the nucleic acidfragment to mediate gene expression or produce a certain phenotype.These terms also refer to modifications of the nucleic acid fragments ofthe instant invention such as deletion or insertion of one or morenucleotides that do not substantially alter the functional properties ofthe resulting nucleic acid fragment relative to the initial, unmodifiedfragment. It is therefore understood, as those skilled in the art willappreciate, that the invention encompasses more than the specificexemplary sequences.

[0098] “Gene” refers to a nucleic acid fragment that expresses aspecific protein, including regulatory sequences preceding (5′non-coding sequences) and following (3′ non-coding sequences) the codingsequence.

[0099] “Native gene” refers to a gene as found in nature with its ownregulatory sequences. In contrast, “chimeric construct” refers to acombination of nucleic acid fragments that are not normally foundtogether in nature. Accordingly, a chimeric construct may compriseregulatory sequences and coding sequences that are derived fromdifferent sources, or regulatory sequences and coding sequences derivedfrom the same source, but arranged in a manner different than thatnormally found in nature. (The term “isolated” means that the sequenceis removed from its natural environment.)

[0100] The term “operably linked” refers to the association of nucleicacid sequences on a single nucleic acid fragment so that the function ofone is regulated by the other. For example, a promoter is operablylinked with a coding sequence when it is capable of regulating theexpression of that coding sequence (i.e., that the coding sequence isunder the transcriptional control of the promoter). Coding sequences canbe operably linked to regulatory sequences in a sense or antisenseorientation.

[0101] The term “expression”, as used herein, refers to the productionof a functional end-product. Expression of a gene involves transcriptionof the gene and translation of the mRNA into a precursor or matureprotein. “Antisense inhibition” refers to the production of antisenseRNA transcripts capable of suppressing the expression of the targetprotein. “Co-suppression” refers to the production of sense RNAtranscripts capable of suppressing the expression of identical orsubstantially similar foreign or endogenous genes (U.S. Pat. No.5,231,020).

[0102] “Mature” protein refers to a post-translationally processedpolypeptide; i.e., one from which any pre- or pro-peptides present inthe primary translation product have been removed. “Precursor” proteinrefers to the primary product of translation of mRNA; i.e., with pre-andpro-peptides still present. Pre- and pro-peptides may be but are notlimited to intracellular localization signals.

[0103] Standard recombinant DNA and molecular cloning techniques usedherein are well known in the art and are described more fully inSambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory Press: Cold SpringHarbor, 1989 (hereinafter “Sambrook”).

[0104] The term “recombinant” refers to an artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques.

[0105] “PCR” or “Polymerase Chain Reaction” is a technique for thesynthesis of large quantities of specific DNA segments, consists of aseries of repetitive cycles (Perkin Elmer Cetus Instruments, Norwalk,Conn.). Typically, the double stranded DNA is heat denatured, the twoprimers complementary to the 3′ boundaries of the target segment areannealed at low temperature and then extended at an intermediatetemperature. One set of these three consecutive steps is referred to asa cycle.

[0106] Polymerase chain reaction (“PCR”) is a powerful technique used toamplify DNA millions of fold, by repeated replication of a template, ina short period of time. (Mullis et al, Cold Spring Harbor Symp. Quant.Biol. 51:263-273 (1986); Erlich et al., European Patent Application50,424; European Patent Application 84,796; European Patent Application258,017, European Patent Application 237,362; Mullis, European PatentApplication 201,184, Mullis et al U.S. Pat. No. 4,683,202; Erlich, U.S.Pat. No. 4,582,788; and Saiki et al, U.S. Pat. No. 4,683,194). Theprocess utilizes sets of specific in vitro synthesized oligonucleotidesto prime DNA synthesis. The design of the primers is dependent upon thesequences of DNA that are desired to be analyzed. The technique iscarried out through many cycles (usually 20-50) of melting the templateat high temperature, allowing the primers to anneal to complementarysequences within the template and then replicating the template with DNApolymerase.

[0107] Furthermore, the present invention also includes a polyclonal ormonoclonal antibody raised against ToxoP30del3C, ToxoP30del4C, orToxoP30MIX1. Such an antibody may be used, for example, in animmunoassay, a vaccine, a kit, or for research purposes.

[0108] As noted above, the present invention also encompasses acomposition or mixture comprising the following two antigens:genetically engineered P30 and P35. This combination or mixture ofantigens may be utilized for the detection of IgG in IgG-positive seraor plasma (i.e., as a diagnostic reagent). Furthermore, the antigens maybe produced either recombinantly or synthetically. Additionally, thepresent invention also includes a composition comprising antibodiesraised against these antigens.

[0109] Further, as noted above, present invention also includes thegenetically engineered P30 antigen. This antigen may be used for thedetection of IgM in IgM-positive sera or plasma (i.e., as a diagnosticreagent), and the antigen may be produced either recombinantly orsynthetically. Furthermore, the present invention also includesantibodies raised against this antigen.

[0110] If, in fact, one wishes to measure both the titer of IgM and IgGin a serum or plasma sample, then a composition or mixture of antigenssuch as genetically engineered P30 and P35 may be utilized in animmunoassay. Such a combination of antigens is also included within thescope of the present invention.

[0111] The present invention also includes methods of detecting IgMand/or IgG using the combinations of antigens described above. Morespecifically, there are two basic types of assays, competitive andnon-competitive (e.g., immunometric and sandwich). In both assays,antibody or antigen reagents are covalently or non-covalently attachedto the solid phase. Linking agents for covalent attachment are known andmay be part of the solid phase or derivatized to it prior to coating.Examples of solid phases used in immunoassays are porous and non-porousmaterials, latex particles, magnetic particles, microparticles, beads,membranes, microtiter wells and plastic tubes. The choice of solid phasematerial and method of labeling the antigen or antibody reagent aredetermined based upon desired assay format performance characteristics.For some immunoassays, no label is required. For example, if the antigenis on a detectable particle such as a red blood cell, reactivity can beestablished based upon agglutination. Alternatively, an antigen-antibodyreaction may result in a visible change (e.g., radial immunodiffusion).In most cases, one of the antibody or antigen reagents used in animmunoassay is attached to a signal generating compound or “label”. Thissignal generating compound or “label” is in itself detectable or may bereacted with one or more additional compounds to generate a detectableproduct (see also U.S. Pat. No. 6,395,472 B1). Examples of such signalgenerating compounds include chromogens, radioisotopes (e.g., 125I,131I, 32P, 3H, 35S, and 14C), fluorescent compounds (e.g., fluorescein,rhodamine), chemiluminescent compounds, particles (visible orfluorescent), nucleic acids, complexing agents, or catalysts such asenzymes (e.g., alkaline phosphatase, acid phosphatase, horseradishperoxidase, beta-galactosidase, and ribonuclease). In the case of enzymeuse, addition of chromo-, fluoro-, or lumo-genic substrate results ingeneration of a detectable signal. Other detection systems such astime-resolved fluorescence, internal-reflection fluorescence,amplification (e.g., polymerase chain reaction) and Raman spectroscopyare also useful.

[0112] There are two general formats commonly used to monitor specificantibody titer and type in humans: (1) antigen is presented on a solidphase, as described above, the human biological fluid containing thespecific antibodies is allowed to react with the antigen, and thenantibody bound to antigen is detected with an anti-human antibodycoupled to a signal generating compound and (2) an anti-human antibodyis bound to the solid phase, the human biological fluid containingspecific antibodies is allowed to react with the bound antibody, andthen antigen attached to a signal generating compound is added to detectspecific antibody present in the fluid sample. In both formats, theanti-human antibody reagent may recognize all antibody classes, oralternatively, be specific for a particular class or subclass ofantibody, depending upon the intended purpose of the assay. These assaysformats as well as other known formats are intended to be within thescope of the present invention and are well known to those of ordinaryskill in the art.

[0113] In particular, two illustrative examples of an immunometricantibody-capture based immunoassay are the IMx Toxo IgM and Toxo IgGantibody assays manufactured by Abbott Laboratories (Abbott Park, Ill.).Both assays are automated Microparticle Enzyme Immunoassays (MEIA) whichmeasure antibodies to Toxoplasma gondii (T. gondii) in human serum orplasma (Safford et al. (1991) J. Clin. Pathol. 44:238-242). One assayqualitatively measures IgM antibodies, indicative of recent exposure oracute infection, and the other assay quantitatively measures IgG,indicative of chronic or past infection. These assays use microparticlescoated with T. gondii antigens as the solid phase. In particular,specimen is added to the coated microparticles to allow antibodiesspecific for T. gondii to bind. Subsequently, an alkaline phosphataseconjugated anti-human IgM (or anti-human IgG) is added that specificallybinds to IgM (or IgG) class antibodies complexed to the T. gondiiantigens. Following addition of a suitable substrate (e.g.,4-methyumbelliferyl phosphate), the rate of enzyme-catalyzed turnover ismonitored based upon fluorescence.

[0114] The mixture of genetically engineered P30 and P35 may be used inthe IgG Abbott immunoassay, and the genetically engineered P30 antigenalone may be utilized in the IgM Abbott immunoassay. Additionally, amixture of genetically engineered P30 and P35 may be utilized in eitherassay, if desired. Furthermore, it must be noted that other non-Abbottassays or platforms may also be utilized, with each antigen orcombination of antigens for purposes of the present invention.

[0115] Thus, the present invention includes a method of detecting IgMantibodies in a test sample comprising the steps of: (a) contacting thetest sample suspected of containing the IgM antibodies with geneticallyengineered P30; (b) detecting the presence of IgM antibodies present inthe test sample. More specifically, the present invention includes amethod of detecting IgM antibodies in a test sample comprising the stepsof: (a) contacting the test sample suspected of containing the IgMantibodies with genetically engineered P30 for a time and underconditions sufficient to allow the formation of IgM antibody/antigencomplexes; (b) adding a conjugate to the resulting IgM antibody/antigencomplexes for a time and under conditions sufficient to allow theconjugate to bind to the bound antibody, the conjugate comprising anantibody (directed against the IgM) attached to a signal generatingcompound capable of generating a detectable signal; (c) detecting thepresence of the IgM antibody which may be present in the test sample bydetecting the signal generated by the signal generating compound. Acontrol or calibrator may also be used which binds to this antigen.Furthermore, the method may also comprise the use of P35 in addition togenetically engineered P30.

[0116] Additionally, the present invention further includes a method fordetecting the presence of IgM which may be present in a test sample.This method comprises the steps of: (a) contacting the test samplesuspected of containing IgM antibodies with anti-antibody specific forthe IgM, for a time and under conditions sufficient to allow forformation of anti-antibody/IgM complexes and (b) detecting the presenceof IgM which may be present in the test sample. (Such anti-antibodiesare commercially available and may be created, for example, byimmunizing a mammal with purified mu-chain of the antibody.)

[0117] More specifically, this method may comprise the steps of: (a)contacting the test sample suspected of containing the IgM antibodieswith anti-antibody specific for the IgM, under time and conditionssufficient to allow the formation of anti-antibody/IgM complexes; (b)adding a conjugate to the resulting anti-antibody/IgM complexes for atime and under conditions sufficient to allow the conjugate to bind tothe bound antibody, the conjugate comprising genetically engineered P30attached to a signal generating compound capable of generating adetectable signal; and (c) detecting the presence of the IgM antibodieswhich may be present in the test sample by detecting the signalgenerated by the signal generating compound. A control or calibrator maybe used which comprises antibody to the anti-antibody. Furthermore, theconjugate may also comprise P35, if desired.

[0118] In each of the above assays, IgG may be detected by substitutingthe genetically engineered P30 with a genetically engineered P30 and P35mixture. Also, anti-antibody specific for IgG will be used.Additionally, if one wishes to detect both IgM and IgG antibodies,genetically engineered P30 and P35 may be utilized in the immunoassay.

[0119] The present invention also encompasses a third method fordetecting the presence of IgM in a test sample. This method comprisesthe steps of: (a) contacting the test sample suspected of containing IgMantibodies with anti-antibody specific for the IgM, under time andconditions sufficient to allow the formation of anti-antibody IgMcomplexes; (b) adding antigen to the resulting anti-antibody/IgMcomplexes for a time and under conditions sufficient to allow theantigen to bind to the bound IgM antibody, the antigen comprising thegenetically engineered P30; and (c) adding a conjugate to the resultinganti-antibody/IgM/antigen complexes, the conjugate comprising acomposition comprising monoclonal or polyclonal antibody attached to asignal generating compound capable of detecting a detectable signal, themonoclonal or polyclonal antibody being directed against the antigen;and (d) detecting the presence of the IgM antibodies which may bepresent in the test sample by detecting the signal generated by thesignal generating compound. Again, a control or calibrator may be usedwhich comprises antibody to the anti-antibody. The antigen mixture mayfurther comprise P35, if desired.

[0120] In this method, IgG may be detected by substituting thegenetically engineered P30 antigen with a genetically engineered P30 andP35 mixture, and utilizing anti-antibody specific for IgG. However, ifone wishes to detect both IgM and IgG antibodies, genetically engineeredP30 and P35 may be utilized in the immunoassay.

[0121] It should also be noted that all of the above methods may be usedto detect IgA antibodies (with an alpha-specific conjugate) and/or IgEantibodies (with an epsilon-specific conjugate) should such detection bedesired.

[0122] Additionally, the present invention also includes a vaccinecomprising a mixture of genetically engineered P30 and P35 antigens anda pharmaceutically acceptable adjuvant. Such a vaccine may beadministered if one desires to raise IgG antibodies in a mammal. Thepresent invention also includes a vaccine comprising the geneticallyengineered P30 antigen and a pharmaceutically acceptable adjuvant (e.g.,Freund's adjuvant or Phosphate Buffered Saline). Such a vaccine may beadministered if one desires to raise IgM antibodies in a mammal.Additionally, the present invention also includes a vaccine comprising amixture of genetically engineered P30 and P35 antigens as well as apharmaceutically acceptable adjuvant. This vaccine should beadministered if one desires to raise both IgM and IgG antibodies in amammal.

[0123] Kits are also included within the scope of the present invention.More specifically, the present invention includes kits for determiningthe presence of IgG and/or IgM. In particular, a kit for determining thepresence of IgM in a test sample comprises a) genetically engineeredP30; and b) a conjugate comprising an antibody (directed against IgM)attached to a signal-generating compound capable of generating adetectable signal. The kit may also contain a control or calibratorwhich comprises a reagent which binds to genetically engineered P30.

[0124] Again, if one desires to detect IgG, rather than IgM, the kitwill comprise a mixture of genetically engineered P30 and P35, ratherthan genetically engineered P30, as well as an antibody directed againstIgG. If one wishes to detect both IgM and IgG, the kit will comprisegenetically engineered P30 and P35.

[0125] The present invention also includes another type of kit fordetecting IgM and/or IgG in a test sample. If utilized for detecting thepresence of IgM, the kit may comprise a) an anti-antibody specific forIgM, and b) genetically engineered P30. A control or calibratorcomprising a reagent which binds to genetically engineered P30 may alsobe included. More specifically, the kit may comprise a) an anti-antibodyspecific for IgM, and b) a conjugate comprising genetically engineeredP30, the conjugate being attached to a signal-generating compoundcapable of generating a detectable signal. Again, the kit may alsocomprise a control or calibrator comprising a reagent which binds togenetically engineered P30.

[0126] Additionally, if one desires to detect IgG, rather than IgM, thekit will comprise a mixture of genetically engineered P30 and P35,rather than genetically engineered P30 alone, as well as anti-antibodyspecific for IgG. If one wishes to detect both IgM and IgG, the kit maycomprise genetically engineered P30 and P35.

[0127] The present invention may be illustrated by the use of thefollowing non-limiting examples:

EXAMPLE 1 General Methodology

[0128] Materials and Sources

[0129] Restriction enzymes, T4 DNA ligase, and the pMAL™ Protein Fusionand Purification System were purchased from New England Biolabs, Inc.(Beverly, Mass.).

[0130] DNA and protein molecular weight standards, plasmid mini-prepkit, ethidium bromide, and pre-cast polyacrylamide gels, were purchasedfrom BioRad Laboratories (Richmond, Calif.).

[0131] Maltose was purchased from Sigma Chemical Co. (St. Louis, Mo.).

[0132] QIAquick PCR Purification Kit and QIAquick Gel Extraction Kitwere purchased from Qiagen, Inc. (Valencia, Calif.).

[0133] Synthetic oligonucleotides were purchased from Sigma Genosys (TheWoodlands, Tex.).

[0134] EPICURIAN Coli™ XL-1 BLUE (recA1 endA1 gyrA96 thi-1 hsdR17 supE44relA1 lac [F′ proAB lacI^(q) ZDM15 Tn10 (Tet^(r))]) supercompetent E.coli cells were obtained from Stratagene Cloning Systems, Inc. (LaJolla, Calif.).

[0135] A GeneAmp™ reagent kit and AmpliTaq DNA Polymerase were purchasedfrom Perkin-Elmer Cetus (Norwalk, Conn.).

[0136] SeaKem GTG agarose was purchased from BioWhittaker MolecularApplications (Rockland, Me.).

[0137] Bacto-Tryptone, Bacto-Yeast Extract, Bacto-Agar ampicillin,buffers, isopropyl-β-D-thiogalactoside (IPTG), bovine serum albumin(BSA), Sephacryl S-300, fetal calf serum (Toxo antibody free), sucrose,sodium azide, urea, EDTA, Triton X-100,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC),2-(N-moropholino)ethanesulfonic acid (MES), inorganic salts, sodiumdodecyl sulfate (SDS), Tween 20, glycerol, 4-methylumbelliferylphosphate (MUP), tris-(hydroxymethyl)aminomethane (Tris), AxSYM Toxo IgGand IgM assay reagents, calibrators, and controls, sulfate-derivatizedmicroparticles, non-fat dry milk, Nipasept, A56620, Brij-35, mouseserum, mannitol, AxSYM instrument, reagents, and commodities werepurchased from Abbott Manufacturing, Inc. (Abbott Park, Ill.).

[0138] Media, Buffers and General Reagents

[0139] “Superbroth II” contained 11.25 g/L tryptone, 22.5 g/L yeastextract, 11.4 g/L potassium phosphate dibasic, 1.7 g/L potassiumphosphate monobasic, 10 ml/L glycerol, adjusted pH to 7.2 with sodiumhydroxide.

[0140] General Methods

[0141] All enzyme digestions of DNA were performed according tosuppliers' instructions. At least 5 units of enzyme were used permicrogram of DNA, and sufficient incubation time was allowed forcomplete digestion of DNA. Supplier protocols were followed for thevarious kits used in the manipulation of DNA and transformation of DNAinto E. coli, for polymerase chain reaction (PCR), and for purificationof maltose binding protein (MBP) and MBP fusion proteins. Standardprocedures were used for preparation of E. coli lysates containingCMP-KDO synthetase (CKS) (U.S. Pat. No. 6,329,157 B1), restrictionanalysis of DNA on agarose gels, purification of DNA fragments fromagarose gels, and ligation of DNA fragments with T4 DNA ligase.(Maniatis et al., Molecular Cloning: A Laboratory Manual, 2^(nd) ed.(Cold Spring Harbor Laboratory Press, New York, 1989)). DNA sequenceanalysis was performed by Lark Technologies, Inc. (Houston, Tex.).

EXAMPLE 2

[0142] Construction of pMBP-ToxoP30 Expression Vectors

[0143] In order to improve the immunoreactivity of the Toxo antigencocktails described in U.S. Pat. No. 6,329,157 B1 in an immunoassay, asuitable heterologous protein expression system was pursued that wouldpermit the production of soluble Toxo P30 in E. coli. The E. colimaltose binding protein (MBP) fusion and purification system describedin U.S. Pat. No. 5,643,758 has been found to be useful for theproduction and purification of soluble fusion proteins in E. coli. (Inparticular, the vectors described therein have sequences coding for therecognition site of a specific protease (e.g., Factor Xa, enterokinaseor Genenase™) such that the protein of interest may be cleaved fromMBP.) Fusion of proteins to MBP enhances their solubility in E. coli(Kapust and Waugh (1999) Protein Science 8, 1668-1674). Severaldifferent constructs were made taking into consideration the observationthat native Toxo P30 is post-translationally cleaved prior to insertioninto the tachyzoite membrane (Burg et al. (1988) J. Immunol.141:3584-3591). The exact cleavage sites for Toxo P30 are unknown.

[0144] Plasmid pToxo-P30 described in U.S. Pat. No. 6,329,157 B1 wasused as template DNA for a series of PCR reactions to generate DNAfragments containing different portions of the Toxo P30 gene. ThepToxo-P30 plasmid DNA was prepared using standard methods. The pMAL-c2X(cytoplasmic expression vector) and pMAL-p2X (periplasmic expressionvector) plasmids were purchased from New England Biolabs, Beverly Mass.and were digested with the restriction enzymes EcoRI and HindIII inpreparation for subcloning the Toxo P30 gene fragments.

[0145] Step A: Construction of pMBP-c2X-ToxoP30(52-336aa)

[0146] The plasmid pMBP-c2X-ToxoP30(52-336aa) was constructed by cloninga DNA fragment containing Toxo P30, obtained by PCR amplification ofToxo P30 DNA contained in plasmid pToxo-P30, into the EcoRI/HindIIIsites of pMAL-c2X (FIG. 1). Plasmid pMAL-c2X was digested withEcoRI/HindIII and the vector backbone was purified with a Qiaquick PCRpurification kit. A sense primer, starting at nucleotide 464 of the P30gene containing an EcoRI site and an antisense primer containing aHindIII site, starting at nucleotide 1318 of the P30 gene (Burg et al.(1988) J. Immunol. 141:3584-3591) were synthesized as shown below:

[0147] Sense Primer [SEQ ID NO:1]5′-GGCGAATTCCTTGTTGCCAATCAAGTTGTCACC-3′ (EcoRI site is underlined)

[0148] Antisense Primer [SEQ ID NO:2]5′-CGCTGAAGCTTTCACGCGACACAAGCTGCGA-3′ (HindIII site is underlined)

[0149] The sense and antisense primers were added to a PCR reactionmixture containing plasmid pToxo-P30. After PCR amplification andpurification of the reaction mixture with a Qiaquick PCR purificationkit, the reaction mixture was digested with EcoRI and HindIII, and the855 base pair DNA fragment containing Toxo P30 was purified with aQiaquick PCR purification kit. The purified 855 base pair fragment wasligated to pMAL-c2X/EcoRI/HindIII overnight at 16° C. The ligationmixture was transformed into competent XL-1 Blue cells. Miniprep DNA wasprepared from the transformants and screened for the presence of the P30DNA sequence by restriction enzyme analysis. PlasmidpMBP-c2X-ToxoP30(52-336aa) contained the Toxo P30 gene cloned at theEcoRI/HindIII sites of pMAL-c2X. The complete DNA sequence [SEQ ID NO:3]of plasmid pMBP-c2X-ToxoP30(52-336aa) is shown in FIG. 2 and thecorresponding amino acid sequence [SEQ ID NO:4] of theMBP-ToxoP30(52-336aa) fusion protein is also shown in FIG. 2, whereinamino acid residues 392-676 of SEQ ID NO:4 correspond to amino acids52-336 of the P30 antigen of Toxoplasma gondii. The DNA sequence [SEQ IDNO:5] of ToxoP30(52-336aa) is shown in FIG. 3, and the correspondingamino acid sequence [SEQ ID NO:6] of the ToxoP30(52-336aa) protein isalso shown in FIG. 3, wherein amino acid residues 1-285 of SEQ ID NO:6correspond to amino acids 52-336 of the P30 antigen of Toxoplasmagondii.

[0150] Step B: Construction of pMBP-p2X-ToxoP30(52-336aa)

[0151] The plasmid pMBP-p2X-ToxoP30(52-336aa) was constructed by cloninga DNA fragment containing Toxo P30, obtained by PCR amplification ofToxo P30 DNA contained in plasmid pToxo-P30, into the EcoRI/HindIIIsites of pMAL-p2X (FIG. 4). Plasmid pMAL-p2X was digested withEcoRI/HindIII, and the vector backbone was purified with a Qiaquick PCRpurification kit. A sense primer, starting at nucleotide 464 of the P30gene containing an EcoRI site, and an antisense primer containing aHindIII site, starting at nucleotide 1318 of the P30 gene (Burg et al.(1988) J. Immunol. 141:3584-3591) were synthesized as shown below:

[0152] Sense Primer [SEQ ID NO:1]5′-GGCGAATTCCTTGTTGCCAATCAAGTTGTCACC-3′ (EcoRI site is underlined.)

[0153] Antisense Primer [SEQ ID NO:2]5′-CGCTGAAGCTTTCACGCGACACAAGCTGCGA-3′ (HindIII site is underlined.)

[0154] The sense and antisense primers were added to a PCR reactionmixture containing plasmid pToxo-P30. After PCR amplification andpurification of the reaction mixture with a Qiaquick PCR purificationkit, the reaction mixture was digested with EcoRI and HindIII, and the855 base pair DNA fragment containing Toxo P30 was purified with aQiaquick PCR purification kit. The purified 855 base pair fragment wasligated to pMAL-p2X/EcoRI/HindIII overnight at 16° C. The ligationmixture was transformed into competent XL-1 Blue cells. Miniprep DNA wasprepared from the transformants and screened for the presence of the P30DNA sequence by restriction enzyme analysis. PlasmidpMBP-p2X-ToxoP30(52-336aa) contained the Toxo P30 gene cloned at theEcoRI/HindIII sites of pMAL-p2X. The complete DNA sequence [SEQ ID NO:7]of plasmid pMBP-p2X-ToxoP30(52-336aa) is shown in FIG. 5, and thecorresponding amino acid sequence [SEQ ID NO:8] of theMBP-ToxoP30P(52-336aa) fusion protein is shown in FIG. 5, wherein aminoacid residues 417-701 of SEQ ID NO:8 correspond to amino acids 52-336 ofthe P30 antigen of Toxoplasma gondii.

[0155] Step C: Construction of pMBP-c2X-ToxoP30del1C(52-324aa)

[0156] The plasmid pMBP-c2X-ToxoP30del1(52-324aa), an intermediate inthe construction of plasmid pMBP-c2X-ToxoP30del1C, was constructed bycloning a DNA fragment containing Toxo P30, obtained by PCRamplification of Toxo P30 DNA contained in plasmid pToxo-P30, into theEcoRI/HindIII sites of pMAL-c2X (FIG. 6). Plasmid pMAL-c2X was digestedwith EcoRI/HindIII and the vector backbone was purified on an agarosegel. A sense primer, starting at nucleotide 464 of the P30 genecontaining an EcoRI site and an antisense primer containing a HindIIIsite, starting at nucleotide 1282 of the P30 gene (Burg et al. (1988) J.Immunol. 141:3584-3591) were synthesized as shown below:

[0157] Sense Primer [SEQ ID NO:1]5′-GGCGAATTCCTTGTTGCCAATCAAGTTGTCACC-3′ (EcoRI site is underlined.)

[0158] Antisense Primer [SEQ ID NO:9]5′-CAGGTCAAGCTTTCACACCATGGCAAAAATGGAAACGTG-3′ (HindIII site isunderlined.)

[0159] The sense and antisense primers were added to a PCR reactionmixture containing plasmid pToxo-P30. After PCR amplification andpurification of the reaction mixture with a Qiaquick PCR purificationkit, the reaction mixture was digested with EcoRI and HindIII, and the819 base pair DNA fragment containing Toxo P30del1 was purified on anagarose gel. The purified 819 base pair fragment was ligated topMAL-c2X/EcoRI/HindIII overnight at 16° C. The ligation mixture wastransformed into competent XL-1 Blue cells. Miniprep DNA was preparedfrom the transformants and screened for the presence of the P30 DNAsequence by restriction enzyme analysis. PlasmidpMBP-c2X-ToxoP30del1(52-324aa) contained the Toxo P30del1 gene cloned atthe EcoRI/HindIII sites of pMAL-c2X.

[0160] Analysis of the DNA sequence of plasmidpMBP-c2X-ToxoP30del1(52-324aa) and analysis of the corresponding aminoacid sequence revealed base changes in the P30 gene resulting in twoamino acid changes from the published sequence (Burg et al. (1988) J.Immunol. 141:3584-3591). These mutations were located downstream of thesynthetic EcoRI site (nucleotide 464) and upstream of a BanI site(nucleotide 1100) following the numbering convention of Burg et al.,cited above. The mutations in plasmid pMBP-c2X-ToxoP30del1(52-324aa)were corrected as follows: Plasmid pMBP-c2X-ToxoP30del1C(52-324aa) wasconstructed by cloning an EcoRI/BanI fragment from plasmidpMBP-p2X-ToxoP30(52-336aa), containing the 5′ corrected portion of theToxo P30 gene, and a BanI/HindIII fragment from plasmidpMBP-c2X-ToxoP30del1(52-324aa), containing the 3′ portion of the ToxoP30del1 gene, into the EcoRI/HindIII sites of pMAL-c2X (FIG. 7). PlasmidpMAL-c2X was digested with EcoRI/HindIII, and the vector backbone waspurified on an agarose gel. Plasmid DNAs pMBP-p2X-ToxoP30(52-336aa) andpMBP-c2X-ToxoP30del1(52-324aa) were prepared by general methods.Plasmids pMBP-p2X-ToxoP30(52-336aa) and pMBP-c2X-ToxoP30del1(52-324aa)were digested with EcoRI/HindIII and the 855 and 819 base pairfragments, containing the Toxo P30 gene, were purified on an agarosegel. The 855 base pair EcoRI/HindIII fragment was digested with BanI andthe 637 base pair EcoRI/BanI fragment, containing the 5′ correctedportion of the Toxo P30 gene, was purified on an agarose gel. The 819base pair EcoRI/HindIII fragment was digested with BanI and the 182 basepair BanI/HindIII fragment, containing the 3′ portion of the ToxoP30del1 gene, was purified on an agarose gel. The purified 637 and 182base pair fragments were ligated to pMAL-c2X/EcoRI/HindIII overnight at16° C. The ligation mixture was transformed into competent XL-1 Bluecells. Miniprep DNA was prepared from the transformants and screened forthe presence of the P30 DNA sequence by restriction enzyme analysis.Plasmid pMBP-c2X-ToxoP30del1C (52-324aa) contained the Toxo P30del1Cgene cloned at the EcoRI/HindIII sites of pMAL-c2X. The complete DNAsequence [SEQ ID NO:10] of plasmid pMBP-c2X-ToxoP30del1C(52-324aa) isshown in FIG. 8 and the corresponding amino acid sequence [SEQ ID NO:11]of the MBP-ToxoP30del1C(52-324aa) fusion protein is also shown in FIG.8, wherein amino acid residues 392-664 of SEQ ID NO:11 correspond toamino acids 52-324 of the P30 antigen of Toxoplasma gondii. The DNAsequence [SEQ ID NO:12] of ToxoP30del1C(52-324aa) is shown in FIG. 9 andthe corresponding amino acid sequence [SEQ ID NO:13] of theToxoP30del1C(52-324aa) protein is also shown in FIG. 9, wherein aminoacid residues 1-273 of SEQ ID NO:13 correspond to amino acids 52-324 ofthe P30 antigen of Toxoplasma gondii.

[0161] Step D: Construction of pMBP-c2X-ToxoP30del2(52-311aa)

[0162] The plasmid pMBP-c2X-ToxoP30del2(52-311aa) was constructed bycloning a DNA fragment containing Toxo P30, obtained by PCRamplification of Toxo P30 DNA contained in plasmid pToxo-P30, into theEcoRI/HindIII sites of pMAL-c2X (FIG. 10). Plasmid pMAL-c2X was digestedwith EcoRI/HindIII and the vector backbone was purified on an agarosegel. A sense primer, starting at nucleotide 464 of the P30 genecontaining an EcoRI site, and an antisense primer containing a HindIIIsite, starting at nucleotide 1243 of the P30 gene (Burg et al. (1988) J.Immunol. 141:3584-3591) were synthesized as shown below:

[0163] Sense Primer [SEQ ID NO:1]5′-GGCGAATTCCTTGTTGCCAATCAAGTTGTCACC-3′ (EcoRI site is underlined.)

[0164] Antisense Primer [SEQ ID NO:14]5′-CAGGTCAAGCTTTCAAGCCGATTTTGCTGACCCTGCAGCCC-3′ (HindIII site isunderlined.)

[0165] The sense and antisense primers were added to a PCR reactionmixture containing plasmid pToxo-P30. After PCR amplification andpurification of the reaction mixture with a Qiaquick PCR purificationkit, the reaction mixture was digested with EcoRI and HindIII, and the780 base pair DNA fragment containing Toxo P30del2 was purified on anagarose gel. The purified 780 base pair fragment was ligated topMAL-c2X/EcoRI/HindIII overnight at 16° C. The ligation mixture wastransformed into competent XL-1 Blue cells. Miniprep DNA was preparedfrom the transformants and screened for the presence of the P30 DNAsequence by restriction enzyme analysis. PlasmidpMBP-c2X-ToxoP30del2(52-311aa) contained the Toxo P30del2 gene cloned atthe EcoRI/HindIII sites of PMAL-c2X. The complete DNA sequence [SEQ IDNO:15] of plasmid pMBP-c2X-ToxoP30del2(52-311aa) is shown in FIG. 11,and the corresponding amino acid sequence [SEQ ID NO:16] of theMBP-ToxoP30del2(52-311aa) fusion protein is also shown in FIG. 11,wherein amino acid residues 392-651 of SEQ ID NO:16 correspond to aminoacids 52-311 of the P30 antigen of Toxoplasma gondii. The DNA sequence[SEQ ID NO:17] of ToxoP30del2(52-311aa) is shown in FIG. 12 and thecorresponding amino acid sequence [SEQ ID NO:18] of theToxoP30del2(52-311aa) protein is also shown in FIG. 12, wherein aminoacid residues 1-260 of SEQ ID NO:18 correspond to amino acids 52-311 ofthe P30 antigen of Toxoplasma gondii.

[0166] Step E: Construction of pMBP-c2X-ToxoP30del3C(52-300aa)

[0167] The plasmid pMBP-c2X-ToxoP30del3(52-300aa), an intermediate inthe construction of plasmid pMBP-c2X-ToxoP30del3C(52-300aa), wasconstructed by cloning a DNA fragment containing Toxo P30, obtained byPCR amplification of Toxo P30 DNA contained in plasmid pToxo-P30, intothe EcoRI/HindIII sites of pMAL-c2X (FIG. 13). Plasmid pMAL-c2X wasdigested with EcoRI/HindIII, and the vector backbone was purified on anagarose gel. A sense primer, starting at nucleotide 464 of the P30 genecontaining an EcoRI site, and an antisense primer containing a HindIIIsite, starting at nucleotide 1210 of the P30 gene (Burg et al. (1988) J.Immunol. 141:3584-3591) were synthesized as shown below:

[0168] Sense Primer [SEQ ID NO:1]5′-GGCGAATTCCTTGTTGCCAATCAAGTTGTCACC-3′ (EcoRI site is underlined.)

[0169] Antisense Primer [SEQ ID NO:19]5′-CAGGTCAAGCTTTCACTCCAGTTTCACGGTACAGTG-3′ (HindIII site is underlined.)

[0170] The sense and antisense primers were added to a PCR reactionmixture containing plasmid pToxo-P30. After PCR amplification andpurification of the reaction mixture with a Qiaquick PCR purificationkit, the reaction mixture was digested with EcoRI and HindIII, and the747 base pair DNA fragment containing Toxo P30del3 was purified on anagarose gel. The purified 747 base pair fragment was ligated topMAL-c2X/EcoRI/HindIII overnight at 16° C. The ligation mixture wastransformed into competent XL-1 Blue cells. Miniprep DNA was preparedfrom the transformants and screened for the presence of the P30 DNAsequence by restriction enzyme analysis. PlasmidpMBP-c2X-ToxoP30del3(52-300aa) contained the Toxo P30del3 gene cloned atthe EcoRI/HindIII sites of pMAL-c2X.

[0171] Analysis of the DNA sequence of plasmidpMBP-c2X-ToxoP30del3(52-300aa) and analysis of the corresponding aminoacid sequence revealed a single base change in the P30 gene thatresulted in the substitution of an amino acid for a stop codon, leadingto premature chain termination of the P30 protein. This mutation waslocated downstream of the synthetic EcoRI site (nucleotide 464) andupstream of a BanI site (nucleotide 1100) following the numberingconvention of Burg et al. The mutation in plasmidpMBP-c2X-ToxoP30del3(52-300aa) was corrected as follows:

[0172] Plasmid pMBP-c2X-ToxoP30del3C(52-300aa) was constructed bycloning an EcoRI/BanI fragment from plasmid pMBP-p2X-ToxoP30(52-336aa),containing the 5′ corrected portion of the Toxo P30 gene, and aBanI/HindIII fragment from plasmid pMBP-c2X-ToxoP30del3(52-300aa),containing the 3′ portion of the Toxo P30del3 gene, into theEcoRI/HindIII sites of pMAL-c2X (FIG. 14). PlasmidpMBP-c2X-ToxoP30del3C(52-300aa) was deposited with the ATCC 10801University Boulevard, Manassas, Va. 20110-2209, under terms of theBudapest Treaty on September ______, 2002, and was accorded AccessionNo. ATCC ______.

[0173] Plasmid pMAL-c2X was digested with EcoRI/HindIII and the vectorbackbone was purified on an agarose gel. Plasmid DNAspMBP-p2X-ToxoP30(52-336aa) and pMBP-c2X-ToxoP30del3(52-300aa) wereprepared by general methods. Plasmids pMBP-p2X-ToxoP30(52-336aa) andpMBP-c2X-ToxoP30del3(52-300aa) were digested with EcoRI/HindIII and the855 and 747 base pair fragments, containing the Toxo P30 gene, werepurified on an agarose gel. The 855 base pair EcoRI/HindIII fragment wasdigested with BanI and the 637 base pair EcoRI/BanI fragment, containingthe 5′ corrected portion of the Toxo P30 gene, was purified on anagarose gel. The 747 base pair EcoRI/HindIII fragment was digested withBanI and the 110 base pair BanI/HindIII fragment, containing the 3′portion of the Toxo P30del3 gene, was purified on an agarose gel. Thepurified 637 and 110 base pair fragments were ligated topMAL-c2X/EcoRI/HindIII overnight at 16° C. The ligation mixture wastransformed into competent XL-1 Blue cells. Miniprep DNA was preparedfrom the transformants and screened for the presence of the P30 DNAsequence by restriction enzyme analysis. Plasmid pMBP-c2X-ToxoP30del3C(52-300aa) contained the ToxoP30del3C gene cloned at the EcoRI/HindIIIsites of pMAL-c2X. The complete DNA sequence [SEQ ID NO:20] of plasmidpMBP-c2X-ToxoP30del3C(52-300aa) is shown in FIG. 15 and thecorresponding amino acid sequence [SEQ ID NO:21] of theMBP-ToxoP30del3C(52-300aa) fusion protein is also shown in FIG. 15,wherein amino acid residues 392-640 of SEQ ID NO:21 correspond to aminoacids 52-300 of the P30 antigen of Toxoplasma gondii. The DNA sequence[SEQ ID NO:22] of ToxoP30del3C(52-300aa) is shown in FIG. 16 and thecorresponding amino acid sequence [SEQ ID NO:23] of theToxoP30del3C(52-300aa) protein is also shown in FIG. 16, wherein aminoacid residues 1-249 of SEQ ID NO:23 correspond to amino acids 52-300 ofthe P30 antigen of Toxoplasma gondii.

[0174] Step F: Construction of pMBP-c2X-ToxoP30del4C(52-294aa)

[0175] The plasmid pMBP-c2X-ToxoP30del4(52-294aa), an intermediate inthe construction of plasmid pMBP-c2X-ToxoP30del4C(52-294aa), wasconstructed by cloning a DNA fragment containing Toxo P30, obtained byPCR amplification of Toxo P30 DNA contained in plasmid p-ToxoP30, intothe EcoRI/HindIII sites of pMAL-c2X (FIG. 17). Plasmid pMAL-c2X wasdigested with EcoRI/HindIII and the vector backbone was purified on anagarose gel. A sense primer, starting at nucleotide 464 of the P30 genecontaining an EcoRI site, and an antisense primer containing a HindIIIsite, starting at nucleotide 1192 of the P30 gene (Burg et al. (1988) J.Immunol. 141:3584-3591) were synthesized as shown below:

[0176] Sense Primer [SEQ ID NO:1]5′-GGCGAATTCCTTGTTGCCAATCAAGTTGTCACC-3′ (EcoRI site is underlined)

[0177] Antisense Primer [SEQ ID NO:24]5′-CAGGTCAAGCTTTCAGTGATGCTTCTCAGGCGATCCCC-3′ (HindIII site isunderlined)

[0178] The sense and antisense primers were added to a PCR reactionmixture containing plasmid pToxo-P30. After PCR amplification andpurification of the reaction mixture with a Qiaquick PCR purificationkit, the reaction mixture was digested with EcoRI and HindIII, and the729 base pair DNA fragment containing Toxo P30del4 was purified on anagarose gel. The purified 729 base pair fragment was ligated topMAL-c2X/EcoRI/HindIII overnight at 16° C. The ligation mixture wastransformed into competent XL-1 Blue cells. Miniprep DNA was preparedfrom the transformants and screened for the presence of the P30 DNAsequence by restriction enzyme analysis. PlasmidpMBP-c2X-ToxoP30del4(52-294aa) contained the Toxo P30del4 gene cloned atthe EcoRI/HindIII sites of pMAL-c2X.

[0179] Analysis of the DNA sequence of plasmidpMBP-c2X-ToxoP30del4(52-294aa) and analysis of the corresponding aminoacid sequence revealed base changes in the P30 gene resulting in twoamino acid changes from the published sequence (Burg et al. (1988) J.Immunol. 141:3584-3591). These mutations were located downstream of thesynthetic EcoRI site (nucleotide 464) and upstream of a BanI site(nucleotide 1100) following the numbering convention of Burg et al. Themutations in plasmid pMBP-c2X-ToxoP30del4(52-294aa) were corrected asfollows:

[0180] Plasmid pMBP-c2X-ToxoP30del4C(52-294aa) was constructed bycloning an EcoRI/BanI fragment from plasmid pMBP-p2X-ToxoP30(52-336aa),containing the 5′ corrected portion of the Toxo P30 gene, and aBanI/HindIII fragment from plasmid pMBP-c2X-ToxoP30del4(52-294aa),containing the 3′ portion of the ToxoP30del4 gene, into theEcoRI/HindIII sites of pMAL-c2X (FIG. 18). PlasmidpMBP-c2X-ToxoP30del4C(52-294aa) was deposited with the ATCC 10801University Boulevard, Manassas, Va. 20110-2209, under terms of theBudapest Treaty on September ______, 2002, and was accorded AccessionNo. ATCC ______.

[0181] Plasmid pMAL-c2X was digested with EcoRI/HindIII and the vectorbackbone was purified on an agarose gel. Plasmid DNAspMBP-p2X-ToxoP30(52-336aa) and pMBP-c2X-ToxoP30del4(52-294aa) wereprepared by general methods. Plasmids pMBP-p2X-ToxoP30(52-336aa) andpMBP-c2X-ToxoP30del4(52-294aa) were digested with EcoRI/HindIII and the855 and 729 base pair fragments, containing the Toxo P30 gene, werepurified on an agarose gel. The 855 base pair EcoRI/HindIII fragment wasdigested with BanI and the 637 base pair EcoRI/BanI fragment, containingthe 5′ corrected portion of the Toxo P30 gene, was purified on anagarose gel. The 729 base pair EcoRI/HindIII fragment was digested withBanI and the 92 base pair BanI/HindIII fragment, containing the 3′portion of the Toxo P30del4 gene and purified on an agarose gel. Thepurified 637 and 92 base pair fragments were ligated topMAL-c2X/EcoRI/HindIII overnight at 16° C. The ligation mixture wastransformed into competent XL-1 Blue cells. Miniprep DNA was preparedfrom the transformants and screened for the presence of the P30 DNAsequence by restriction enzyme analysis. Plasmid pMBP-c2X-ToxoP30del4C(52-294aa) contained the Toxo P30del4C gene cloned at the EcoRI/HindIIIsites of pMAL-c2X. The complete DNA sequence [SEQ ID NO:25] of plasmidpMBP-c2X-ToxoP30del4C(52-294aa) is shown in FIG. 19 and thecorresponding amino acid sequence [SEQ ID NO:26] of theMBP-ToxoP30del4C(52-294aa) fusion protein is also shown in FIG. 19,wherein amino acid residues 392-634 of SEQ ID NO:26 correspond to aminoacids 52-294 of the P30 antigen of Toxoplasma gondii. The DNA sequence[SEQ ID NO:27] of ToxoP30del4C(52-294aa) is shown in FIG. 20 and thecorresponding amino acid sequence [SEQ ID NO:28] of theToxoP30del4C(52-294aa) protein is also shown in FIG. 20, wherein aminoacid residues 1-243 of SEQ ID NO:28 correspond to amino acids 52-294 ofthe P30 antigen of Toxoplasma gondii.

[0182] Step G: Construction of pMBP-c2X-ToxoP30del4del8(83-294aa)

[0183] The plasmid pMBP-c2X-ToxoP30del4del8(83-294aa) was constructed bycloning a DNA fragment containing Toxo P30, obtained by PCRamplification of Toxo P30 DNA contained in plasmid pToxo-P30, into theEcoRI/HindIII sites of pMAL-c2X (FIG. 21). Plasmid pMAL-c2X was digestedwith EcoRI/HindIII and the vector backbone was purified on an agarosegel. A sense primer, starting at nucleotide 557 of the P30 genecontaining an EcoRI site, and an antisense primer containing a HindIIIsite, starting at nucleotide 1192 of the P30 gene (Burg et al. (1988) J.Immunol. 141:3584-3591) were synthesized as shown below:

[0184] Sense Primer [SEQ ID NO:29] 5′-GGCGAATTCCCTAAAACAGCGCTCACAGAG-3′(EcoRI site is underlined.)

[0185] Antisense Primer [SEQ ID NO:24]5′-CAGGTCAAGCTTTCAGTGATGCTTCTCAGGCGATCCCC-3′ (HindIII site isunderlined.)

[0186] The sense and antisense primers were added to a PCR reactionmixture containing plasmid pToxo-P30. After PCR amplification andpurification of the reaction mixture with a Qiaquick PCR purificationkit, the reaction mixture was digested with EcoRI and HindIII, and the636 base pair DNA fragment containing Toxo P30del4del8 was purified onan agarose gel. The purified 636 base pair fragment was ligated topMAL-c2X/EcoRI/HindIII overnight at 16° C. The ligation mixture wastransformed into competent XL-1 Blue cells. Miniprep DNA was preparedfrom the transformants and screened for the presence of the P30 DNAsequence by restriction enzyme analysis. PlasmidpMBP-c2X-ToxoP30del4del8(83-294aa) contained the ToxoP30del4del8 genecloned at the EcoRI/HindIII sites of pMAL-c2X. The complete DNA sequence[SEQ ID NO:30] of plasmid pMBP-c2X-ToxoP30del4del8(83-294aa) is shown inFIG. 22 and the corresponding amino acid sequence [SEQ ID NO:31] of theMBP-ToxoP30del4del8(83-294aa) fusion protein is also shown in FIG. 22,wherein amino acid residues 392-603 of SEQ ID NO:31 correspond to aminoacids 83-294 of the P30 antigen of Toxoplasma gondii. The DNA sequence[SEQ ID NO:32] of ToxoP30del4del8(83-294aa) is shown in FIG. 23 and thecorresponding amino acid sequence [SEQ ID NO:33] of theToxoP30del4del8(83-294aa) protein is also shown in FIG. 23, whereinamino acid residues 1-212 of SEQ ID NO:33 correspond to amino acids83-294 of the P30 antigen of Toxoplasma gondii.

[0187] Step H: Construction of pMBP-c2X-ToxoP30del10(52-284aa)

[0188] The plasmid pMBP-c2X-ToxoP30del10(52-284aa) was constructed bycloning a DNA fragment containing Toxo P30, obtained by PCRamplification of Toxo P30 DNA contained in plasmid pToxo-P30, into theEcoRI/HindIII sites of pMAL-c2X (FIG. 24). Plasmid pMAL-c2X was digestedwith EcoRI/HindIII and the vector backbone was purified on an agarosegel. A sense primer, starting at nucleotide 464 of the P30 genecontaining an EcoRI site, and an antisense primer containing a HindIIIsite, starting at nucleotide 1162 of the P30 gene (Burg et al. (1988) J.Immunol. 141:3584-3591) were synthesized as shown below:

[0189] Sense Primer [SEQ ID NO:1]5′-GGCGAATTCCTTGTTGCCAATCAAGTTGTCACC-3′ (EcoRI site is underlined.)

[0190] Antisense Primer [SEQ ID NO:34]5′-CAGGTCAAGCTTTCATCCAATAATGACGCTTTTTGACTC-3′ (HindIII site isunderlined.)

[0191] The sense and antisense primers were added to a PCR reactionmixture containing plasmid pToxo-P30. After PCR amplification andpurification of the reaction mixture with a Qiaquick PCR purificationkit, the reaction mixture was digested with EcoRI and HindIII, and the699 base pair DNA fragment containing Toxo P30del10 was purified on anagarose gel. The purified 699 base pair fragment was ligated topMAL-c2X/EcoRI/HindIII overnight at 16° C. The ligation mixture wastransformed into competent XL-1 Blue cells. Miniprep DNA was preparedfrom the transformants and screened for the presence of the P30 DNAsequence by restriction enzyme analysis. PlasmidpMBP-c2X-ToxoP30del10(52-284aa) contained the Toxo P30del10 gene clonedat the EcoRI/HindIII sites of pMAL-c2X. The complete DNA sequence [SEQID NO:35] of plasmid pMBP-c2X-ToxoP30del10(52-284aa) is shown in FIG. 25and the corresponding amino acid sequence [SEQ ID NO:36] of theMBP-ToxoP30del10(52-284aa) fusion protein is also shown in FIG. 25,wherein amino acid residues 392-624 of SEQ ID NO:36 correspond to aminoacids 52-284 of the P30 antigen of Toxoplasma gondii, with the exceptionthat amino acid residue 546 of SEQ ID NO:36 is glycine instead ofglutamic acid. The DNA sequence [SEQ ID NO:37] of ToxoP30del10(52-284aa)is shown in FIG. 26 and the corresponding amino acid sequence [SEQ IDNO:38] of the ToxoP30del10(52-284aa) protein is also shown in FIG. 26,wherein amino acid residues 1-233 of SEQ ID NO:38 correspond to aminoacids 52-284 of the P30 antigen of Toxoplasma gondii, with the exceptionthat amino acid residue 155 of SEQ ID NO:38 is glycine instead ofglutamic acid.

[0192] Step I: Construction of pMBP-c2X-ToxoP30del11(52-214aa)

[0193] The plasmid pMBP-c2X-ToxoP30del11(52-214aa) was constructed bycloning a DNA fragment containing Toxo P30, obtained by PCRamplification of Toxo P30 DNA contained in plasmid pToxo-P30, into theEcoRI/HindIII sites of pMAL-c2X (FIG. 27). Plasmid pMAL-c2X was digestedwith EcoRI/HindIII and the vector backbone was purified on an agarosegel. A sense primer, starting at nucleotide 464 of the P30 genecontaining an EcoRI site, and an antisense primer containing a HindIIIsite, starting at nucleotide 952 of the P30 gene (Burg et al. (1988) J.Immunol. 141:3584-3591) were synthesized as shown below:

[0194] Sense Primer [SEQ ID NO:1]5′-GGCGAATTCCTTGTTGCCAATCAAGTTGTCACC-3′ (EcoRI site is underlined.)

[0195] Antisense Primer [SEQ ID NO:39]5′-CAGGTCAAGCTTTCACACGAGGGTCATTGTAGTGGG-3′ (HindIII site is underlined.)

[0196] The sense and antisense primers were added to a PCR reactionmixture containing plasmid pToxo-P30. After PCR amplification andpurification of the reaction mixture with a Qiaquick PCR purificationkit, the reaction mixture was digested with EcoRI and HindIII, and the489 base pair DNA fragment containing Toxo P30del11 was purified on anagarose gel. The purified 489 base pair fragment was ligated topMAL-c2X/EcoRI/HindIII overnight at 16° C. The ligation mixture wastransformed into competent XL-1 Blue cells. Miniprep DNA was preparedfrom the transformants and screened for the presence of the P30 DNAsequence by restriction enzyme analysis. PlasmidpMBP-c2X-ToxoP30del11(52-214aa) contained the Toxo P30del11 gene clonedat the EcoRI/HindIII sites of pMAL-c2X. The complete DNA sequence [SEQID NO:40] of plasmid pMBP-c2X-ToxoP30del11(52-214aa) is shown in FIG. 28and the corresponding amino acid sequence [SEQ ID NO:41] of theMBP-ToxoP30del11(52-214aa) fusion protein is also shown in FIG. 28,wherein amino acid residues 392-554 of SEQ ID NO:41 correspond to aminoacids 52-214 of the P30 antigen of Toxoplasma gondii. The DNA sequence[SEQ ID NO:42] of ToxoP30del11(52-214aa) is shown in FIG. 29 and thecorresponding amino acid sequence [SEQ ID NO:43] of theToxoP30del11(52-214aa) protein is also shown in FIG. 29, wherein aminoacid residues 1-163 of SEQ ID NO:43 correspond to amino acids 52-214 ofthe P30 antigen of Toxoplasma gondii.

EXAMPLE 3 Expression of rpMBP-c2X-ToxoP30 Antigens in E. coli

[0197] Step A: Expression of Cloned Genes in E. coli

[0198] Bacterial clones pMBP-c2X-ToxoP30(52-336aa),pMBP-c2X-ToxoP30del1C(52-324aa), pMBP-c2X-ToxoP30del2(52-311aa),pMBP-c2X-ToxoP30del3C(52-300aa), pMBP-c2X-ToxoP30del4C(52-294aa),pMBP-c2X-ToxoP30del4del8(83-294aa), pMBP-c2X-ToxoP30del10(52-284aa) andpMBP-c2X-ToxoP30del11(52-214aa) expressing the MBP fusion proteinsrpMBP-c2X-ToxoP30(52-336aa), rpMBP-c2X-ToxoP30del1C(52-324aa),rpMBP-c2X-ToxoP30del2(52-311aa), rpMBP-c2X-ToxoP30del3C(52-300aa),rpMBP-c2X-ToxoP30del4C(52-294aa), rpMBP-c2X-ToxoP30del4del8(83-294aa),rpMBP-c2X-ToxoP30del10(52-284aa) and rpMBP-c2X-ToxoP30del11(52-214aa) ofExample 2 were grown in “SUPERBROTH II” media containing 100 μg/mlampicillin to log phase, and the synthesis of the MBP-ToxoP30 fusionproteins was induced by the addition of IPTG as previously described(Robinson et al. (1993) J. Clin. Microbiol. 31:629-635). After 4 hourspost-induction, the cells were harvested, and the cell pellets werestored at −80° C. until protein purification occurred.

[0199] Step B: Purification of MBP-ToxoP30 Fusion Proteins

[0200] Soluble fusion proteins rpMBP-c2X-ToxoP30(52-336aa),rpMBP-c2X-ToxoP30del1C(52-324aa), rpMBP-c2X-ToxoP30del2(52-311aa),rpMBP-c2X-ToxoP30del3C(52-300aa), rpMBP-c2X-ToxoP30del4C(52-294aa),rpMBP-c2X-ToxoP30del4del8(83-294aa), rpMBP-c2X-ToxoP30del10(52-284aa)and rpMBP-c2X-ToxoP30del11(52-214aa) were purified after lysis from cellpaste following the New England Biolabs pMAL Protein Fusion andPurification instruction manual. Following lysis and centrifugation, thecrude supernatants containing the fusion proteins were loaded onto anamylose affinity column. Following washing of the column, the fusionproteins were eluted from the column with maltose, appropriate columnfractions were pooled and filtered through a 0.2μ filter, and thenstored at 2-8° C. until coating onto microparticles.

EXAMPLE 4 Evaluation of rpMBP-c2X-ToxoP30 Antigens in an Automated ToxoIgG and IgM Immunoassay

[0201] Step A: Coating of rpMBP-c2X-ToxoP30 Antigens onto Microparticles

[0202] Prior to coating microparticles, the rpMBP-c2X-ToxoP30(52-336aa),rpMBP-c2X-ToxoP30del1C(52-324aa), rpMBP-c2X-ToxoP30del2(52-311aa),rpMBP-c2X-ToxoP30del3C(52-300aa), rpMBP-c2X-ToxoP30del4C(52-294aa),rpMBP-c2X-ToxoP30del4del8(83-294aa), rpMBP-c2X-ToxoP30del10(52-284aa)and rpMBP-c2X-ToxoP30del11(52-214aa) antigens were diluted to aconcentration of 1 mg/ml and incubated at 37° C. for 24 hours. Followingthe heat treatment step, the rpMBP-c2X-ToxoP30 antigens were coatedseparately onto sulfate-derivatized polystyrene microparticles (1-5%solids) in a vessel containing MES pH 6.2 buffer with EDAC for 30minutes at room temperature, on an end over end rotator. The coatedmicroparticles were then collected by centrifugation at 14,000×g for 10minutes, and the supernatant was discarded. The microparticles wereresuspended in a microparticle storage buffer containing Tris buffer, pH7.5, EDTA, sodium chloride, Tween 20, fetal calf serum (Toxo antibodyfree), sodium azide, and sucrose using a syringe and needle. Themicroparticles were then diluted with microparticle storage buffer to afinal concentration of 0.1-0.3% solids and filled into plastic bottles.

[0203] Step B: Description of the AxSYM® Toxo IgG v2 Reagent Pack,Calibrators, Controls, Panel 6, and Assay Diskette

[0204] The reagent pack for the automated AxSYM Toxo IgG v2 assay isdesigned for the detection of human anti-Toxo IgG and consists of thefollowing components. Bottle number one contains microparticles coatedwith purified recombinant Toxo antigens (Example 4A) in a microparticlestorage buffer. In order to prevent human anti-MBP or anti-CKSantibodies causing a false positive reaction in the assay, purified MBPor CKS can be added to the microparticle storage buffer. Bottle numbertwo contains the preferred conjugate, a goat anti-human IgG alkalinephosphatase conjugate. This conjugate is titered to determine a workingconcentration of 0.1-5 μg/ml. The conjugate is diluted into a conjugatediluent containing Tris buffer, pH 7.4, sodium, calcium, magnesium, andzinc chloride, Nipasept, A56620, non-fat dry milk, Brij-35, mouse serum,and mannitol. Bottle number three contains the preferred assay diluentto minimize non-specific binding to the microparticles and assay matrix.This assay diluent consists of a Tris buffer, pH 7.5 containing sodiumchloride, sodium EDTA, non-fat dry milk, Nipasept, A56620, and Tween 20.Bottle number four contains a phosphate buffer line diluent.

[0205] Six Assay Calibrators labeled A-F are derived from Toxo IgGpositive plasma pools or human anti-Toxo IgG monoclonal antibodies andare required to calibrate the AxSYM® Toxo IgG v2 assay. Thesecalibrators are matched to calibrators previously matched to the TOX-SWHO International Standard. The concentration range of these calibratorsis 0-300 IU/ml. Positive and Negative controls are required to evaluatethe assay calibration and establish assay validity. The Positive Controlis prepared from Toxo IgG positive plasma pools or human anti-Toxo IgGmonoclonal antibodies. The Negative Control is prepared from Toxo IgGnegative plasma pools. Panel 6 is a pool of Toxo IgG and IgM positiveplasma units derived from blood donors with an acute toxoplasmosis.

[0206] The assay diskette for the AxSYM® Toxo IgG v2 assay contains theassay protocol software necessary to run the automated immunoassay onthe Abbott “AxSYM®” instrument (Abbott Laboratories, Abbott Park, Ill.).In addition to the AxSYM® Toxo IgG v2 Reagent Pack, assay Calibrators,and Controls described above, the following assay components located onthe instrument are required to run the assay: Sample Cups, AxSYM® LineDiluent, MEIA buffer, Reaction Vessels, MUP, and Matrix Cells. Thesequence of events for the automated assay are as follows: The pipettingprobe in the kitting center delivers the patient sample and line diluentto the reaction vessel sample well; the pipetting probe then kits theappropriate volumes of assay diluent, line diluent, and conjugaterequired for the assay from the reagent pack into the designatedreaction vessel wells; this probe then delivers the recombinant Toxoantigen coated microparticles from the reagent pack and an aliquot ofthe diluted sample to the designated reaction vessel well; the reactionvessel is then transferred to the process carousel; Toxo-specificantibodies bind to the Toxo recombinant antigen coated microparticlesforming an antigen-antibody complex; assay diluent is added to thereaction mixture and matrix cell and then an aliquot of the reactionmixture is transferred to the glass fiber matrix in the auxiliarycarousel; the microparticles bind irreversibly to the matrix; the matrixis washed with MEIA buffer and line diluent to remove unboundantibodies; goat anti-human IgG alkaline phosphate conjugate is added tothe matrix and binds to the Toxo-specific IgG captured by the Toxorecombinant antigens; the matrix is then washed with MEIA buffer toremove any unbound enzyme-antibody conjugate; the enzyme substrate MUPis added to the matrix; the alkaline phosphatase enzyme present on thematrix attached to the goat anti-human IgG catalyzes the hydrolysis ofthe phosphoryl moiety from MUP, producing a highly fluorescent productwhich is measured by the AxSYM MEIA optical system; the signal intensity(rate counts) is proportional to the amount of Toxo-specific IgGantibodies present in the sample.

[0207] Step C: Description of the AxSYM Toxo® IgM v2 Reagent Pack, IndexCalibrator, Controls, and Assay Diskette

[0208] The reagent pack for the automated AxSYM Toxo IgM v2 assay isdesigned for the detection of human anti-Toxo IgM and consists of thefollowing components. Bottle number one contains microparticles coatedwith purified recombinant Toxo antigens (Example 4A) in a microparticlestorage buffer. In order to prevent human anti-MBP or anti-CKSantibodies causing a false positive reaction in the assay, purified MBPor CKS can be added to the microparticle storage buffer. Bottle numbertwo contains the preferred conjugate, a goat anti-human IgM alkalinephosphatase conjugate. This conjugate is titered to determine a workingconcentration of 0.1-5 μg/ml. The conjugate is diluted into a conjugatediluent containing Tris buffer, pH 7.4, sodium, calcium, magnesium, andzinc chloride, Nipasept, A56620, non-fat dry milk, Brij-35, mouse serum,and mannitol. Bottle number three contains the preferred assay diluentto minimize non-specific binding to the microparticles and assay matrix.This assay diluent consists of a Tris buffer, pH 7.5 containing sodiumchloride, sodium EDTA, non-fat dry milk, Nipasept, A56620, and Tween 20.Bottle number four contains either phosphate buffer line diluent or RFNeutralization Buffer.

[0209] The Index Calibrator is derived from Toxo IgM positive plasmapools or human anti-Toxo IgM monoclonal antibodies and is required tocalibrate the AxSYM® Toxo IgM v2 assay. Positive and Negative controlsare required to evaluate the assay calibration and establish assayvalidity. The Positive Control is prepared from Toxo IgM positive plasmapools or human anti-Toxo IgM monoclonal antibodies. The Negative Controlis prepared from Toxo IgM negative plasma pools.

[0210] The assay diskette for the AxSYM® Toxo IgM v2 assay contains theassay protocol software necessary to run the automated immunoassay onthe Abbott “AxSYM” instrument (Abbott Laboratories, Abbott Park, Ill.).In addition to the AxSYM® Toxo IgM v2 Reagent Pack, Index Calibrator,and Controls described above, the following assay components located onthe instrument are required to run the assay: Sample Cups, AxSYM® LineDiluent, MEIA buffer, Reaction Vessels, MUP, and Matrix Cells. Thesequence of events for the automated assay are as follows: The pipettingprobe in the kitting center delivers the patient sample and line diluentto the reaction vessel sample well; the pipetting probe then kits theappropriate volumes of assay diluent, line diluent or RF NeutralizationBuffer, and conjugate required for the assay from the reagent pack intothe designated reaction vessel wells; this probe then delivers therecombinant Toxo antigen coated microparticles from the reagent pack andan aliquot of the diluted sample to the designated reaction vessel well;the reaction vessel is then transferred to the process carousel;Toxo-specific antibodies bind to the Toxo recombinant antigen coatedmicroparticles forming an antigen-antibody complex; assay diluent isadded to the reaction mixture and matrix cell and then an aliquot of thereaction mixture is transferred to the glass fiber matrix in theauxiliary carousel; the microparticles bind irreversibly to the matrix;the matrix is washed with MEIA buffer and line diluent or RFNeutralization Buffer to remove unbound antibodies; goat anti-human IgMalkaline phosphate conjugate is added to the matrix and binds to theToxo-specific IgM captured by the Toxo recombinant antigens; the matrixis then washed with MEIA buffer to remove any unbound enzyme-antibodyconjugate; the enzyme substrate MUP is added to the matrix; the alkalinephosphatase enzyme present on the matrix attached to the goat anti-humanIgM catalyzes the hydrolysis of the phosphoryl moiety from MUP,producing a highly fluorescent product which is measured by the AxSYM®MEIA optical system; the signal intensity (rate counts) is proportionalto the amount of Toxo-specific IgM antibodies present in the sample.

[0211] Step D: Evaluation of MBP fusion proteinsrpMBP-c2X-ToxoP30(52-336aa), rpMBP-c2X-ToxoP30del1C(52-324aa),rpMBP-c2X-ToxoP30del2(52-311aa), rpMBP-c2X-ToxoP30del3C(52-300aa),rpMBP-c2X-ToxoP30del4C(52-294aa), rpMBP-c2X-ToxoP30del4del8(83-294aa),rpMBP-c2X-ToxoP30del10(52-284aa) and rpMBP-c2X-ToxoP30del11(52-214aa) inthe AxSYM® Toxo TgG v2 and Toxo IgM v2 Immunoassays

[0212] The AxSYM® Toxo IgG and IgM reagent packs were assembled asdescribed in Examples 4B and 4C using the microparticles coated with theToxo antigens described in Example 4A. The Toxo IgG A and F calibrators(Acal and Fcal) and Panel 6 (PNL6) were tested with the AxSYM Toxo IgGv2 reagent packs and the Toxo IgM Negative Control (NC), IndexCalibrator (IC), and Panel 6 (PNL6) were tested with the AxSYM® Toxo IgMv2 reagent packs. The results are shown below in Tables 1 and 2. TABLE 1Evaluation of the rpMBP-c2X-ToxoP30 Antigens in the AxSYM ® Toxo IgG v2Assay Rate Counts PNL6 Fcal/ / Antigen Coated Acal Fcal Acal PNL6 AcalrpMBP-c2X-ToxoP30 40 3495 87  643 16 (52-336aa) rpMBP-c2X-ToxoP30 292599 90  544 19 del1C(52-324aa) rpMBP-c2X-ToxoP30 28 3191 114 1129 40del2(52-311aa) rpMBP-c2X-ToxoP30 29 3199 110 1112 38 del3C(52-300aa)rpMBP-c2X-ToxoP30 29 3352 116 1302 45 del4C(52-294aa) rpMBP-c2X-ToxoP3040 4277 107 1285 32 del10(52-284aa) rpMBP-c2X-ToxoP30 48 4076 85 1118 23del11(52-214aa) rpMBP-c2X-ToxoP30 34  59 1.7  37 1.1 del4del8(83-294aa)

[0213] TABLE 2 Evaluation of the rpMBP-c2X-ToxoP30 Antigens in theAxSYM® Toxo IgM v2 Assay Rate Counts PNL6/ Antigen Coated NC IC PNL6 NCrpMBP-c2X-ToxoP30 33 175 210 6.4 (52-336aa) rpMBP-c2X-ToxoP30del1C 48189 250 5.2 (52-324aa) rpMBP-c2X-ToxoP30del2 43 315 440 10.2 (52-311aa)rpMBP-c2X-ToxoP30del3C 49 187 459 9.4 (52-300aa) rpMBP-c2X-ToxoP30del4C51 203 527 10.3 (52-294aa) rpMBP-c2X-ToxoP30del10 39 158 370 9.5(52-284aa) rpMBP-c2X-ToxoP30del11 36 130 312 8.7 (52-214aa)rpMBP-c2X-ToxoP30del4 38 75 38 1.0 del8 (83-294aa)

[0214] As can be seen in both Tables 1 and 2, a surprising result wasobtained. In particular, deletion of amino acids from the C-terminus ofthe ToxoP30 antigen resulted in improved Toxo-specific IgG and IgMimmunoreactivity, as measured by increased rate counts for Panel 6 andimproved rate count ratios for Fcal/Acal, Panel 6/Acal, and Panel 6/NC,up to a deletion of 42 amino acids (compare proteinrpMBP-c2X-ToxoP30del4C(52-294aa) with proteinrpMBP-c2X-ToxoP30(52-336aa) in Tables 1 and 2). The geneticallyengineered rpMBP-c2X-ToxoP30del4C(52-294aa) protein yielded maximal ratecounts for Panel 6 and maximal rate count ratios in both assays. Theseresults suggest that small deletions of the C-terminus of ToxoP30 revealor make available new epitopes for binding of Toxo-specific IgG and IgMthat are occluded in the full-length protein. Deletion of additionalC-terminal amino acids (compare protein rpMBP-c2X-ToxoP30del10(52-284aa)and rpMBP-c2X-ToxoP30del11(52-214aa) with proteinrpMBP-c2X-ToxoP30del4C(52-294aa) in Tables 1 and 2) resulted in reducedimmunoreactivity, suggesting the loss of IgG and IgM epitopes withlarger C-terminal deletions. In contrast, the introduction of a small 30amino acid deletion in the N-terminus of the optimal proteinrpMBP-c2X-ToxoP30del4C, which generated the proteinrpMBP-c2X-ToxoP30del4del8(83-294aa), completely abolished Toxo-specificIgG and IgM immunoreactivity. These results also suggest that some ofthe cysteine residues present in the C-terminal portion of the ToxoP30protein are dispensable for optimal Toxo IgG and IgM immunoreactivity.For example, the optimal protein rpMBP-c2X-ToxoP30del4C(52-294aa)contains 11 cysteine residues and the proteinrpMBP-c2X-ToxoP30del11(52-214aa), which demonstrated good but notoptimal immunoreactivity, contains 7 cysteine residues.

EXAMPLE 5 Construction of Toxo P30 Synthetic Genes Containing MutationsWhich Change Cysteine Residues to Alanine

[0215] Based on the results obtained from deletion analysis of the ToxoP30 gene in Example 4D, a new series of MBP-ToxoP30 fusion proteins wasconstructed. Since there are 12 cysteine residues present in the matureToxo P30 protein (Burg et al. (1988) J. Immunol. 141:3584-3591;Velge-Roussel et al. (1994) Molec. Biochem. Parasitol. 66:31-38), thereare mathematically 2¹² or 4,096 different Toxo P30 proteins that can beconstructed which contain various combinations of changing one or moreof the twelve cysteine residues to alanine. It would certainly beimpossible to try all 4,096 different cysteine to alanine combinationsto further optimize the immunoreactivity of the Toxo P30 antigen. Hence,the results in Example 4D were used to narrow the number of differentmutant Toxo P30 genes to build that have the potential for improved ToxoIgG and IgM immunoreactivity in an automated immunoassay. Mutantoligonucleotides were designed for the in vitro synthesis of three ToxoP30 genes that contain mutations which change various cysteine residuesto alanine, and also introduce the same 3′ deletion in the Toxo P30 genepresent in ToxoP30del3C(52-300aa) (SEQ ID NO:22 and FIG. 16).

[0216] The synthesis of each Toxo P30 gene required the synthesis andassembly of 16 overlapping oligonucleotides. These oligonucleotidesranged from 67-72 bases in length with neighboring oligonucleotidesoverlapping by 20-23 residues. The P30 genes were assembled by recursivePCR followed by PCR amplification of the assembled genes(Withers-Martinez et al. (1999) Protein Engr. 12:1113-1120; Prytulla etal. (1996) FEBS Lett. 399:283-289; Kataoka et al. (1998) Biochem.Biophys. Res. Comm. 250:409-413) using a P30 sense primer containing anEcoRI site and an antisense primer containing a HindIII site. After PCRamplification, the P30 gene was digested with EcoRI and HindIII,purified on an agarose gel, and ligated to the pMAL-c2X vector backbonewhich had been digested by EcoRI and HindIII as shown schematically inFIG. 30.

[0217] Step A: Construction of pMBP-c2X-ToxoP30MIX1

[0218] The plasmid pMBP-c2X-ToxoP30MIX1 was constructed by cloning asynthetic DNA fragment containing Toxo P30, obtained by the synthesisand assembly of 16 oligonucleotides, into the EcoRI/HindIII sites ofpMAL-c2X (FIG. 30). This plasmid differs from plasmidpMBP-c2X-ToxoP30del3C(52-300aa) of EXAMPLE 2E in that the Toxo P30 DNAsequence in plasmid pMBP-c2X-ToxoP30MIX1 has been changed so that fiveof the twelve cysteine residues of Toxo P30 (cysteine nos. 8-12) havebeen changed to alanine. Plasmid pMAL-c2X was digested withEcoRI/HindIII and the vector backbone was purified on an agarose gel.The following oligonucleotides were synthesized for construction of theToxoP30MIX1 gene: P30.001 5′-CTTGTTGCCAATCAAGTTGTCACCTGCCCAGAT [SEQ IDNO:44] AAAAAATCGACAGCCGCGGTCATTCTCACACCGACG G-3′ P30.0025′-GAGGCTCTGTGAGCGCTGTTTTAGGGCACTTGA [SEQ ID NO:45]GAGTGAAGTGGTTCTCCGTCGGTGTGAGAATGACCG -3′ P30.0035′-CCTAAAACAGCGCTCACAGAGCCTCCCACTCTT [SEQ ID NO:46]GCGTACTCACCCAACAGGCAAATCTGCCCAGCGG -3′ P30.0045′-GGAATCAAGGAGCTCAATGTTACAGCCTTTGAT [SEQ ID NO:47]GTACAGCTACTTGTAGTACCCGCTGGGCAGATTTGC CTC-3′ P30.0055′-GTAACATTGAGCTCCTTGATTCCTGAAGCAGAA [SEQ ID NO:48]GATAGCTGGTGGACGGGGGATTCTGCTAGTCTCGAC ACGG-3′ P30.0065′-CTGCGTTGTCACGGGGAACTTCTCGATTGGAAC [SEQ ID NO:49]TGTGAGTTTGATGCCTGCCGTGTCGAGACTAGCAGA ATC-3′ P30.0075′-GAAGTTCCCCGTGACAACGCAGACGTTTGTGGT [SEQ ID NO:50]CGGTTGCATCAAGGGAGACGACGCACAGAGTTGTAT G-3′ P30.0085′-GCGACATTATTGACGACCGATGAGGCTCTGGCT [SEQ ID NO:51]TGTACTGTCACCGTGACCATACAACTCTGTGCGTCG TC-3′ P30.0095′-CATCGGTCGTCAATAATGTCGCAAGGTGCTCCT [SEQ ID NO:52]ACGGTGCAGACAGCACTCTTGGTCCTGTCAAGTTGT C-3′ P30.010Ala85′-GACTCCATCTTTCCCAGCCACGAGGGTCATTGT [SEQ ID NO:53]AGTGGGTCCTTCCGCAGACAACTTGACAGGACCAAG AG-3′ P30.011Ala8Ala95′-GTGGCTGGGAAAGATGGAGTCAAAGTTCCTCAA [SEQ ID NO:54]GACAACAATCAGTACGCTTCCGGGACGACGCTGACT GG-3′ P30.012Ala9Ala105′-GTTCTCAGTTAATTTTGGCAAAATATCTTTGAA [SEQ ID NO:55]CGATTTCTCGTTAGCACCAGTCAGCGTCGTCCCGGA AG-3′ P30.0135′-GATATTTTGCCAAAATTAACTGAGAACCCGTGG [SEQ ID NO:56]CAGGGTAACGCTTCGAGTGATAAGGGTGCCACGCTA AC-3′ P30.0145′-CCAATAATGACGCTTTTTGACTCGGCTGGAAAT [SEQ ID NO:57]GCTTCCTTCTTGATCGTTAGCGTGGCACCCTTATCA C-3′ P30.015Ala11Ala125′-GTCAAAAAGCGTCATTATTGGAGCTACAGGGGG [SEQ ID NO:58]ATCGCCTGAGAAGCATCACGCTACCGTGAAACTGGA C-3′ P30.01GAla125′-GACTGGCTGTTCCCGCAGCCGATTTTGCTGACC [SEQ ID NO:59]CTGCAGCCCCGGCAAACTCCAGTTTCACGGTAGCGT G-3′

[0219] In the first step of gene synthesis, 4 picomoles of eacholigonucleotide were mixed together and assembled using recursive PCR asfollows: 1 cycle at 95° C. for 5 minutes followed by 35 cycles at 95° C.for 1 minute, 55° C. for 2 minutes, 72° C. for 2 minutes; 1 cycle at 72°C. for 10 minutes followed by a soak cycle at 4° C. In the second stepof gene synthesis, a sense primer, starting at nucleotide 464 of the P30gene containing an EcoRI site, and an antisense primer containing aHindIII site, starting at nucleotide 1210 of the P30 gene (Burg et al.(1988) J. Immunol. 141:3584-3591) were synthesized as shown below:

[0220] Sense Primer [SEQ ID NO:1]5′-GGCGAATTCCTTGTTGCCAATCAAGTTGTCACC-3′ (EcoRI site is underlined.)

[0221] Antisense Primer [SEQ ID NO:60]5′-CAGGTCAAGCTTTCACTCCAGTTTCACGGTAGCGTG-3′ (HindIII site is underlined.)

[0222] The sense and antisense primers were added to a PCR reactionmixture containing the assembled oligonucleotides from the first step ofgene synthesis. After PCR amplification and purification of the reactionmixture with a Qiaquick PCR purification kit, the reaction mixture wasdigested with EcoRI and HindIII, and the 747 base pair DNA fragmentcontaining Toxo P30MIX1 was purified on an agarose gel. The purified 747base pair fragment was ligated to pMAL-c2X/EcoRI/HindIII overnight at16° C. The ligation mixture was transformed into competent XL-1 Bluecells. Miniprep DNA was prepared from the transformants and screened forthe presence of the P30 synthetic DNA sequence by restriction enzymeanalysis. Plasmid pMBP-c2X-ToxoP30MIX1 contained the Toxo P30MIX1 genecloned at the EcoRI/HindIII sites of pMAL-c2X. The complete DNA sequence[SEQ ID NO:61] of plasmid pMBP-c2X-ToxoP30MIX1 is shown in FIG. 31, andthe corresponding amino acid sequence [SEQ ID NO:62] of theMBP-ToxoP30MIX1 fusion protein is also shown in FIG. 31, whereincysteine amino acid residues located at 555, 570, 578, 625, 635 of SEQID NO:21 are now alanine amino acids located at 555, 570, 578, 625, 635of SEQ ID NO:62. Plasmid pMBP-c2X-ToxoP30MIX1 was deposited with theATCC 10801 University Boulevard, Manassas, Va. 20110-2209, under termsof the Budapest Treaty on September ______, 2002, and was accordedAccession No. ATCC ______. The DNA sequence [SEQ ID NO:63] ofToxoP30MIX1 is shown in FIG. 32, and the corresponding amino acidsequence [SEQ ID NO:64] of the ToxoP30MIX1 protein is also shown in FIG.32, wherein cysteine amino acid residues located at 164, 179, 187, 234,244 of SEQ ID NO:23 are now alanine amino acids located at 164, 179,187, 234, 244 of SEQ ID NO:64.

[0223] Step B: Construction of pMBP-c2X-ToxoP30MIX3

[0224] The plasmid pMBP-c2X-ToxoP30MIX3 was constructed by cloning asynthetic DNA fragment containing Toxo P30, obtained by the synthesisand assembly of 16 oligonucleotides, into the EcoRI/HindIII sites ofpMAL-c2X (FIG. 30). This plasmid differs from plasmidpMBP-c2X-ToxoP30del3C(52-300aa) of EXAMPLE 2E in that the Toxo P30 DNAsequence in plasmid pMBP-c2X-ToxoP30MIX3 has been changed so that six ofthe twelve cysteine residues of Toxo P30 (cysteine nos. 7-12) have beenchanged to alanine. Plasmid pMAL-c2X was digested with EcoRI/HindIII,and the vector backbone was purified on an agarose gel. The followingoligonucleotides were synthesized for construction of the ToxoP30MIX3gene: P30.001 5′-CTTGTTGCCAATCAAGTTGTCACCTGCCCAGAT [SEQ ID NO:44]AAAAAATCGACAGCCGCGGTCATTCTCACACCGACG G-3′ P30.0025′-GAGGCTCTGTGAGCGCTGTTTTAGGGCACTTGA [SEQ ID NO:45]GAGTGAAGTGGTTCTCCGTCGGTGTGAGAATGACCG -3′ P30.0035′-CCTAAAACAGCGCTCACAGAGCCTCCCACTCTT [SEQ ID NO:46]GCGTACTCACCCAACAGGCAAATCTGCCCAGCGG -3′ P30.0045′-GGAATCAAGGAGCTCAATGTTACAGCCTTTGAT [SEQ ID NO:47]GTACAGCTACTTGTAGTACCCGCTGGGCAGATTTGC CTC-3′ P30.0055′-GTAACATTGAGCTCCTTGATTCCTGAAGCAGAA [SEQ ID NO:48]GATAGCTGGTGGACGGGGGATTCTGCTAGTCTCGAC ACGG-3′ P30.0065′-CTGCGTTGTCACGGGGAACTTCTCGATTGGAAC [SEQ ID NO:49]TGTGAGTTTGATGCCTGCCGTGTCGAGACTAGCAGA ATC-3′ P30.0075′-GAAGTTCCCCGTGACAACGCAGACGTTTGTGGT [SEQ ID NO:50]CGGTTGCATCAAGGGAGACGACGCACAGAGTTGTAT G-3′ P30.0085′-GCGACATTATTGACGACCGATGAGGCTCTGGCT [SEQ ID NO:51]TGTACTGTCACCGTGACCATACAACTCTGTGCGTCG TC-3′ P30.009Ala75′-CATCGGTCGTCAATAATGTCGCAAGGGCTTCCT [SEQ ID NO:65]ACGGTGCAGACAGCACTCTTGGTCCTGTCAAGTTGT C-3′ P30.010Ala85′-GACTCCATCTTTCCCAGCCACGAGGGTCATTGT [SEQ ID NO:53]AGTGGGTCCTTCCGCAGACAACTTGACAGGACCAAG AG-3′ P30.011Ala8Ala95′-GTGGCTGGGAAAGATGGAGTCAAAGTTCCTCAA [SEQ ID NO:54]GACAACAATCAGTACGCTTCCGGGACGACGCTGACT GG-3′ P30.012Ala9Ala105′-GTTCTCAGTTAATTTTGGCAAAATATCTTTGAA [SEQ ID NO:55]CGATTTCTCGTTAGCACCAGTCAGCGTCGTCCCGGA AG-3′ P30.0135′-GATATTTTGCCAAAATTAACTGAGAACCCGTGG [SEQ ID NO:56]CAGGGTAACGCTTCGAGTGATAAGGGTGCCACGCTA AC-3′ P30.0145′-CCAATAATGACGCTTTTTGACTCGGCTGGAAAT [SEQ ID NO:57]GCTTCCTTCTTGATCGTTAGCGTGGCACCCTTATCA C-3′ P30.015Ala11Ala125′-GTCAAAAAGCGTCATTATTGGAGCTACAGGGGG [SEQ ID NO:58]ATCGCCTGAGAAGCATCACGCTACCGTGAAACTGGA G-3′ P30.016A1a125′-GACTGGCTGTTCCCGCAGCCGATTTTGCTGACCC [SEQ ID NO:59]TGCAGCCCCGGCAAACTCCAGTTTCACGGTAGCGTG -3′

[0225] In the first step of gene synthesis, 4 picomoles of eacholigonucleotide were mixed together and assembled using recursive PCR asfollows: 1 cycle at 95° C. for 5 minutes followed by 35 cycles at 95° C.for 1 minute, 55° C. for 2 minutes, 72° C. for 2 minutes; 1 cycle at 72°C. for 10 minutes followed by a soak cycle at 4° C. In the second stepof gene synthesis, a sense primer, starting at nucleotide 464 of the P30gene containing an EcoRI site, and an antisense primer containing aHindIII site, starting at nucleotide 1210 of the P30 gene (Burg et al.(1988) J. Immunol. 141:3584-3591) were synthesized as shown below:

[0226] Sense Primer [SEQ ID NO:1]5′-GGCGAATTCCTTGTTGCCAATCAAGTTGTCACC-3′ (EcoRI site is underlined.)

[0227] Antisense Primer [SEQ ID NO:60]5′-CAGGTCAAGCTTTCACTCCAGTTTCACGGTAGCGTG-3′ (HindilI site is underlined.)

[0228] The sense and antisense primers were added to a PCR reactionmixture containing the assembled oligonucleotides from the first step ofgene synthesis. After PCR amplification and purification of the reactionmixture with a Qiaquick PCR purification kit, the reaction mixture wasdigested with EcoRI and HindIII, and the 747 base pair DNA fragmentcontaining Toxo P30MIX3 was purified on an agarose gel. The purified 747base pair fragment was ligated to pMAL-c2X/EcoRI/HindIII overnight at16° C. The ligation mixture was transformed into competent XL-1 Bluecells. Miniprep DNA was prepared from the transformants and screened forthe presence of the P30 synthetic DNA sequence by restriction enzymeanalysis. Plasmid pMBP-c2X-ToxoP30MIX3 contained the Toxo P30MIX3 genecloned at the EcoRI/HindIII sites of pMAL-c2X. The complete DNA sequence[SEQ ID NO:66] of plasmid pMBP-c2X-ToxoP30MIX3 is shown in FIG. 33, andthe corresponding amino acid sequence [SEQ ID NO:67] of theMBP-ToxoP30MIX3 fusion protein is also shown in FIG. 33, whereincysteine amino acid residues located at 530, 555, 570, 578, 625, 635 ofSEQ ID NO:21 are now alanine amino acids located at 530, 555, 570, 578,625, 635 of SEQ ID NO:67. The DNA sequence [SEQ ID NO:68] of ToxoP30MIX3is shown in FIG. 34, and the corresponding amino acid sequence [SEQ IDNO:69] of the ToxoP30MIX3 protein is also shown in FIG. 34, whereincysteine amino acid residues located at 139, 164, 179, 187, 234, 244 ofSEQ ID NO:23 are now alanine amino acids located at 139, 164, 179, 187,234, 244 of SEQ ID NO:69.

[0229] Step C: Construction of pMBP-c2X-ToxoP30MIX5

[0230] The plasmid pMBP-c2X-ToxoP30MIX5 was constructed by cloning asynthetic DNA fragment containing Toxo P30, obtained by the synthesisand assembly of 16 oligonucleotides, into the EcoRI/HindIII sites ofpMAL-c2X (FIG. 30). This plasmid differs from plasmidpMBP-c2X-ToxoP30del3C(52-300aa) of EXAMPLE 2E in that the Toxo P30 DNAsequence in plasmid pMBP-c2X-ToxoP30MIX5 has been changed so that six ofthe twelve cysteine residues of Toxo P30 (cysteine nos. 2 and 8-12) havebeen changed to alanine. Plasmid pMAL-c2X was digested withEcoRI/HindIII and the vector backbone was purified on an agarose gel.The following oligonucleotides were synthesized for construction of theToxoP30MIX5 gene: P30.001 5′-CTTGTTGCCAATCAAGTTGTCACCTGCCCACAT [SEQ IDNO:44] AAAAAATCGACAGCCGCGGTCATTCTCACACCGACG G-3′ P30.002Ala25′-GAGGCTCTGTGAGCGCTGTTTTAGGAGCCTTGA [SEQ ID NO:70]GAGTGAAGTGGTTCTCCGTCGGTGTGAGAATGACCG -3′ P30.0035′-CCTAAAACAGCGCTCACAGAGCCTCCCACTCTT [SEQ ID NO:46]GCGTACTCACCCAACAGGCAAATCTGCCCAGCGG -3′ P30.0045′-GGAATCAAGGAGCTCAATGTTACAGCCTTTGAT [SEQ ID NO:47]GTACAGCTACTTGTAGTACCCGCTGGGCAGATTTGC CTG-3′ P30.0055′-GTAACATTGAGCTCCTTGATTCCTGAAGCAGAA [SEQ ID NO:48]GATAGCTGGTGGACGGGGGATTCTGCTAGTCTCGAC ACGG-3′ P30.0065′-CTGCGTTGTCACGGGGAACTTCTCGATTGGAAC [SEQ ID NO:49]TGTGAGTTTGATGCCTGCCGTGTCGAGACTAGCAGA ATC-3′ P30.0075′-GAAGTTCCCCGTGACAACGCAGACGTTTGTGGT [SEQ ID NO:50]CGGTTGCATCAAGGGAGACGACGCACAGAGTTGTAT G-3′ P30.0085′-GCGACATTATTGACGACCGATGAGGCTCTGGCT [SEQ ID NO:51]TGTACTGTCACCGTGACCATACAACTCTGTGCGTCG TC-3′ P30.0095′-CATCGGTCGTCAATAATGTCGCAAGGTGCTCCT [SEQ ID NO:52]ACGGTGCAGACAGCACTCTTGGTCCTGTCAAGTTGT C-3′ P30.010Ala85′-GACTCCATCTTTCCCAGCCACGAGGGTCATTGT [SEQ ID NO:53]AGTGGGTCCTTCCGCAGACAACTTGACAGGACCAAG AG-3′ P30.011Ala8Ala95′-GTGGCTGGGAAAGATGGAGTCAAAGTTCCTCAA [SEQ ID NO:54]GACAACAATCAGTACGCTTCCGGGACGACGCTGACT GG-3′ P30.012Ala9Ala105′-GTTCTCAGTTAATTTTGGCAAAATATCTTTGAA [SEQ ID NO:55]CGATTTCTCGTTAGCACCAGTCAGCGTCGTCCCGGA AG-3′ P30.0135′-GATATTTTGCCAAAATTAACTGAGAACCCGTGG [SEQ ID NO:56]CAGGGTAACGCTTCGAGTGATAAGGGTGCCACGCTA AC-3′ P30.0145′-CCAATAATGACGCTTTTTGACTCGGCTGGAAAT [SEQ ID NO:57]GCTTCCTTCTTGATCGTTAGCGTGGCACCCTTATCA C-3′ P30.015Ala11Ala125′-GTCAAAAAGCGTCATTATTGGAGCTACAGGGGG [SEQ ID NO:58]ATCGCCTGAGAAGCATCACGCTACCGTGAAACTGGA G-3′ P30.016Ala125′-GACTGGCTGTTCCCGCAGCCGATTTTGCTGACC [SEQ ID NO:59]CTGCAGCCCCGGCAAACTCCAGTTTCACGGTAGCGT G-3′

[0231] In the first step of gene synthesis, 4 picomoles of eacholigonucleotide were mixed together and assembled using recursive PCR asfollows: 1 cycle at 95° C. for 5 minutes followed by 35 cycles at 95° C.for 1 minute, 55° C. for 2 minutes, 72° C. for 2 minutes; 1 cycle at 72°C. for 10 minutes followed by a soak cycle at 4° C. In the second stepof gene synthesis, a sense primer, starting at nucleotide 464 of the P30gene containing an EcoRI site, and an antisense primer containing aHindIII site, starting at nucleotide 1210 of the P30 gene (Burg et al.(1988) J. Immunol. 141:3584-3591) were synthesized as shown below:

[0232] Sense Primer [SEQ ID NO:1]5′-GGCGAATTCCTTGTTGCCAATCAAGTTGTCACC-3′ (EcoRI site is underlined.)

[0233] Antisense Primer [SEQ ID NO:60]5′-CAGGTCAAGCTTTCACTCCAGTTTCACGGTAGCGTG-3′ (HindIII site is underlined.)

[0234] The sense and antisense primers were added to a PCR reactionmixture containing the assembled oligonucleotides from the first step ofgene synthesis. After PCR amplification and purification of the reactionmixture with a Qiaquick PCR purification kit, the reaction mixture wasdigested with EcoRI and HindIII, and the 747 base pair DNA fragmentcontaining Toxo P30MIX5 was purified on an agarose gel. The purified 747base pair fragment was ligated to pMAL-c2X/EcoRI/HindIII overnight at16° C. The ligation mixture was transformed into competent XL-1 Bluecells. Miniprep DNA was prepared from the transformants and screened forthe presence of the P30 synthetic DNA sequence by restriction enzymeanalysis. Plasmid pMBP-c2X-ToxoP30MIX5 contained the Toxo P30MIX5 genecloned at the EcoRI/HindIII sites of pMAL-c2X. The complete DNA sequence[SEQ ID NO:71] of plasmid pMBP-c2X-ToxoP30MIX5 is shown in FIG. 35, andthe corresponding amino acid sequence [SEQ ID NO:72] of theMBP-ToxoP30MIX5 fusion protein is also shown in FIG. 35, whereincysteine amino acid residues located at 422, 555, 570, 578, 625, 635 ofSEQ ID NO:21 are now alanine amino acids located at 422, 555, 570, 578,625, 635 of SEQ ID NO:72. The DNA sequence [SEQ ID NO:73] of ToxoP30MIX5is shown in FIG. 36, and the corresponding amino acid sequence [SEQ IDNO:74] of the ToxoP30MIX5 protein is also shown in FIG. 36, whereincysteine amino acid residues located at 31, 164, 179, 187, 234, 244 ofSEQ ID NO:23 are now alanine amino acids located at 31, 164, 179, 187,234, 244 of SEQ ID NO:74.

EXAMPLE 6 Expression of rpMBP-c2X-ToxoP30MIX Antigens in E. coli

[0235] Step A: Expression of Cloned Genes in E. coli

[0236] Bacterial clones pMBP-c2X-ToxoP30MTX1, pMBP-c2X-ToxoP30MIX3, andpMBP-c2X-ToxoP30MIX5 expressing the MBP fusion proteinsrpMBP-c2X-ToxoP30MIX1, rpMBP-c2X-ToxoP30MIX3, and rpMBP-c2X-ToxoP30MIX5of Example 5 were grown in “SUPERBROTH II” media containing 100 μg/mlampicillin to log phase, and the synthesis of the MBP-ToxoP30MIX fusionproteins was induced by the addition of IPTG as previously described(Robinson et al. (1993) J. Clin. Microbiol. 31:629-635). After 4 hourspost-induction, the cells were harvested, and the cell pellets werestored at −80° C. until protein purification occurred.

[0237] Step B: Purification of MBP-ToxoP30MIX Fusion Proteins

[0238] Soluble fusion proteins rpMBP-c2X-ToxoP30MIX1,rpMBP-c2X-ToxoP30MIX3, and rpMBP-c2X-ToxoP30MIX5 were purified afterlysis from cell paste following the New England Biolabs pMAL ProteinFusion and Purification instruction manual. Following lysis andcentrifugation, the crude supernatants containing the fusion proteinswere loaded onto an amylose affinity column. Following washing of thecolumn, the fusion proteins were eluted from the column with maltose,appropriate column fractions were pooled and filtered through a 0.2μfilter, and then stored at 2-8° C. until coating onto microparticles.

EXAMPLE 7 Evaluation of rpMBP-c2X-ToxoP30MIX Antigens in an AutomatedToxo IgG and IgM Immunoassay

[0239] Step A: Coating of rpMBP-c2X-ToxoP30MIX Antigens ontoMicroparticles

[0240] Prior to coating microparticles, the rpMBP-c2X-ToxoP30MIX1,rpMBP-c2X-ToxoP30MIX3 and rpMBP-c2X-ToxoP30MIX5 antigens were diluted toa concentration of 1 mg/ml and incubated at 37° C. for 24 hours.Following the heat treatment step, the rpMBP-c2X-ToxoP30MIX antigenswere coated separately onto sulfate-derivatized polystyrenemicroparticles (1-5% solids) in a vessel containing MES pH 6.2 bufferwith EDAC for 30 minutes at room temperature, on an end over endrotator. The coated microparticles were then collected by centrifugationat 14,000×g for 10 minutes and the supernatant was discarded. Themicroparticles were resuspended in a microparticle storage buffercontaining Tris buffer, pH 7.5, EDTA, sodium chloride, Tween 20, fetalcalf serum (Toxo antibody free), sodium azide, and sucrose using asyringe and needle. The microparticles were then diluted withmicroparticle storage buffer to a final concentration of 0.1-0.3% solidsand filled into plastic bottles.

[0241] Step B: Evaluation of MBP fusion proteins rpMBP-c2X-ToxoP30MIX1,rpMBP-c2X-ToxoP30MIX3 and rpMBP-c2X-ToxoP30MIX5 in the AxSYM Toxo IgG v2and Toxo IgM v2 Immunoassays

[0242] The AXSYM® Toxo IgG and IgM reagent packs were assembled asdescribed in Examples 4B and 4C using the microparticles coated with theToxo antigens described in Example 7A. The Toxo IgG A and F calibrators(Acal and Fcal) and Panel 6 (PNL6) were tested with the AxSYM Toxo IgGv2 reagent packs and the Toxo IgM Negative Control (NC), IndexCalibrator (IC), and Panel 6 (PNL6) were tested with the AxSYM Toxo IgMv2 reagent packs. The results are shown below in Tables 3 and 4. TABLE 3Evaluation of the rpMBP-c2X-ToxoP30MIX Antigens in the AxSYM Toxo IgG v2Assay Rate Counts Fcal/ PNL6/ Antigen Coated Acal Fcal Acal PNL6 AcalrpMBP-c2X-ToxoP30 34 3847 113 1445 43 MIX1 (Ala8-12) rpMBP-c2X-ToxoP3035 3704 106 1043 30 MIX3 (Ala7-12) rpMBP-c2X-Toxop30 35  57 1.6  23 0.7MIX5 (Ala2, Ala8-12)

[0243] TABLE 4 Evaluation of the rpMBP-c2X-ToxoP30MIX Antigens in theAxSYM Toxo IgM v2 Assay Rate Counts PNL6/ Antigen Coated NC IC PNL6 NCrpMBP-c2X-ToxoP30 37 149 459 12.4 MIX1 (Ala8-12) rpMBP-c2X-ToxoP30 44111 368 8.4 MIX3 (Ala7-12) rpMBP-c2X-ToxoP30 31 49 27 0.9 MIX5 (Ala2,Ala8-12)

[0244] As can be seen in Tables 3 and 4, the genetically engineeredrpMBP-c2X-ToxoP30MIX1 antigen, which contained five C-terminal cysteineresidues substituted with alanine, yielded the best Toxo IgG and IgMimmunoreactivity as measured by the highest rate counts for Panel 6 andhighest rate count ratios for Fcal/Acal, Panel 6/Acal, and Panel 6/NC.In addition, the rpMBP-c2X-ToxoP30MIX1 antigen yielded the highest ratecounts for Panel6 and the highest Panel6/NC rate count ratio in the ToxoIgM v2 assay for any rpMBP-c2X-ToxoP30 antigen tested (see Tables 1 and2). The rpMBP-c2X-ToxoP30MIX5 antigen, which contained five C-terminalcysteine residues substituted with alanine residues plus thesubstitution of cysteine no. 2 with alanine, was not immunoreactive ineither assay. This result was consistent with the results obtained withthe rpMBP-c2X-ToxoP30del4del8(83-294aa) antigen (see Tables 1 and 2), inwhich deletion of the first two cysteine residues of Toxo P30 resultedin complete loss of immunoreactivity. Thus, the surprising result wasobtained that substitution of several cysteine residues in theC-terminal half of Toxo P30 with alanine results in superior Toxo IgGand IgM immunoreactivity of the antigen whereas substitution of a singlecysteine with alanine in the N-terminal half of Toxo P30 completelyabolishes immunoreactivity.

EXAMPLE 8 Purification and Coating of the rpToxoP35S Antigen

[0245] The rpToxoP35S antigen described in U.S. Pat. No. 6,329,157 B1was expressed in E. coli, and cell paste was harvested as described inExample 6A. This antigen was then purified from cell paste and coatedonto microparticles as described below.

[0246] Step A: Purification of the rpToxoP35S Antigen

[0247] The rpToxoP35S antigen was expressed in E. coli as an insolublefusion protein. Following lysis of the cell paste, the inclusion bodiescontaining the rpToxoP35S antigen were washed with water, phosphatebuffer, Triton X-100, and urea. The rpToxoP35S antigen was thensolubilized in a SDS/DTT buffer and applied to a Sephacryl S-300 column.The appropriate column fractions were pooled, diluted to a concentrationof 1 mg/ml and filtered through a 0.2μ filter, and then stored at −80°C. until coating.

[0248] Step B: Coating of the rpToxoP35S Antigen onto Microparticles

[0249] The rpToxoP35S antigen was thawed and brought into solution bymild warming followed by centrifugation to remove particulate matter.This antigen was then dialyzed against three changes of MES buffer, pH6.2 at room temperature overnight and then coated ontosulfate-derivatized polystyrene microparticles (1-5% solids) in a vesselcontaining MES pH 6.2 buffer with EDAC for 30 minutes at roomtemperature, on an end over end rotator. The coated microparticles werethen collected by centrifugation at 14,000×g for 10 minutes and thesupernatant was discarded. The microparticles were resuspended in amicroparticle storage buffer containing Tris buffer, pH 7.5, EDTA,sodium chloride, Tween 20, fetal calf serum (Toxo antibody free), sodiumazide, and sucrose using a syringe and needle. The microparticles werethen diluted with microparticle storage buffer to a final concentrationof 0.1-0.3% solids and filled into plastic bottles.

EXAMPLE 9 Development of Antigen Cocktails Employing the GeneticallyEngineered P30 Antigens

[0250] After achieving a significant improvement in the Toxo IgG and IgMimmunoreactivity of the P30 antigen through genetic engineering(Examples 4 and 7), a preliminary re-evaluation of microparticles coatedwith the Toxo antigens described in U.S. Pat. No. 6,329,157 B1 in theAxSYM® Toxo IgG v2 and IgM v2 assays was performed. Evaluation of theseantigen coated microparticles in conjunction with microparticles coatedwith the P30 antigens rpMBP-c2X-ToxoP30del3C(52-300aa),rpMBP-c2X-ToxoP30del4C(52-294aa), and rpMBP-c2X-ToxoP30MIX1 suggestedthat a new combination of either the rpMBP-c2X-ToxoP30del3C(52-300aa),rpMBP-c2X-ToxoP30del4C(52-294aa), or rpMBP-c2X-ToxoP30MIX1 antigen withthe rpToxoP35S antigen could improve the performance of the AXSYM® ToxoIgG v2 assay. In addition, the rpMBP-c2X-ToxoP30del3C(52-300aa),rpMBP-c2X-ToxoP30del4C(52-294aa), or rpMBP-c2X-ToxoP30MIX1 antigensalone could improve the performance of the AxSYM® Toxo IgM v2 assay. Inorder to demonstrate the diagnostic utility of the geneticallyengineered P30 antigens and the new genetically engineered P30/P35antigen cocktail, human sera negative for Toxo antibodies and serasourced from patients with an acute or chronic toxoplasmosis were testedin the AxSYM® Toxo IgG v2 and IgM v2 assays as described below.

[0251] Step A: Assembly of AxSYM® Toxo IgG v2 Reagent Packs

[0252] Purified Toxo antigens rpMBP-c2X-ToxoP30del3C(52-300aa),rpMBP-c2X-ToxoP30del4C(52-294aa), rpMBP-c2X-ToxoP30MIX1, and rpToxoP35Swere coated unto microparticles as previously described in Examples 4Aand 7A. The microparticles were then diluted to a final concentration0.2% solids and the following three microparticle blends were made: 2:1v/v blend of rpMBP-c2X-ToxoP30del3C(52-300aa):rpToxoP35S coatedmicroparticles (labeled as P30del3/P35); 2:1 v/v blend ofrpMBP-c2X-ToxoP30del4C(52-294aa):rpToxoP35S coated microparticles(labeled as P30del4/P35); and a 2:1 v/v blend ofrpMBP-c2X-ToxoP30MIX1:rpToxoP35S coated microparticles (labeled asP30MIX1/P35). These three microparticle blends were filled into plasticbottles, assembled into individual AxSYM® Toxo IgG v2 reagent kits asdescribed in Example 4B, and labeled as P30del3C/35, P30del4C/P35, andP30MIX1/P35.

[0253] Step B: Assembly of AxSYM® Toxo IgM v2 Reagent Packs

[0254] Purified Toxo antigens rpMBP-c2X-ToxoP30del3C(52-300aa),rpMBP-c2X-ToxoP30del4C(52-294aa), rpMBP-c2X-ToxoP30MIX1, and rpToxoP35Swere coated onto microparticles as previously described in Examples 4Aand 7A. The microparticles were then diluted to a final concentration of0.2% solids and filled into plastic bottles. AxSYM® Toxo IgM v2 kitswere assembled with each coated microparticle as described in Example 4Cand labeled as P30del3C, P30del4C, and P30MIX1.

[0255] Step C: Human sera for Testing

[0256] Three groups of human sera from a French population were testedin this evaluation: Group 1 (n=100) human sera negative for Toxo IgG andIgM antibodies by the Abbott IMx® Toxo IgG and IgM assays, respectively(Abbott Laboratories, Abbott Park, Ill.); Group 2 (n=56) human serapositive for Toxo IgG and negative for Toxo IgM antibodies by the AbbottIMx® Toxo IgG and IgM assays, respectively; Group 3 (n=52) human serapositive for Toxo IgG antibodies by a high sensitivity directagglutination assay (HSDA) (Desmonts, G. and Remington, J. S. (1980) J.Clin. Microbiol. 11:562-568) and positive for Toxo IgM antibodies by anIgM immunocapture assay (IC-M) (Pouletty et al. (1985) J. Immunol.Methods 76:289-298). The assay calibrators and controls for the AxSYM®Toxo IgG v2 and Toxo IgM v2 assays were run as previously described inExamples 4B-D. The Abbott AxSYM® Toxo IgG and IgM assays (AbbottLaboratories Abbott Park, Ill.), which use the tachyzoite antigen fordetection of Toxo-specific IgG and IgM, were included as referenceassays during specimen testing.

[0257] Step D: Evaluation of the AxSYM® Toxo IgG v2 Assays

[0258] All specimens in Groups 1-3 were tested by the AxSYM® Toxo IgG v2assays (P30del3C/35, P30del4C/P35, and P30MIX1/P35) and by the AxSYM®Toxo IgG assay. The same assay cutoff of 3 IU/ml for the AxSYM Toxo IgGassay was employed for AxSYM® Toxo IgG v2 assays, with an equivocal zonefrom 2-3 IU/ml. The performance of the recombinant antigen based AxSYM®Toxo IgG v2 assays was compared to the tachyzoite antigen based AxSYM®Toxo IgG assay and is shown in Tables 5-7. TABLE 5 Evaluation of theAxSYM ® Toxo IgG v2 P30del3C/P35 assay

[0259] TABLE 6 Evaluation of the AxSYM ® Toxo IgG v2 P30del14C/P35 assay

[0260] TABLE 7 Evaluation of the AxSYM ® Toxo IgG v2 P30MIX1/P35 assay

[0261] As can be seen from Tables 5-7, the AxSYM® Toxo IgG v2 assayusing the combination of a genetically engineered antigen P30 antigen(P30del3C, P30del4C, or P30MIX1) with the P35 antigen is both asensitive and specific assay for the detection of Toxoplasma-specificIgG as demonstrated by the overall high relative diagnostic sensitivity(100%), specificity (100%), and agreement (100%). All three AxSYM® ToxoIgG v2 assays were in excellent agreement quantitatively with the AxSYM®Toxo IgG assay, as measured by the correlation coefficients, all ofwhich were 0.95 or greater. The Toxo recombinant antigen cocktailcomprised of the genetically engineered Toxo P30 antigen (P30del3C,P30del4C, or P30MIX1) and the P35 antigen, in combination with theAxSYM® Toxo IgG v2 assay, is both necessary and sufficient to replacethe tachyzoite for the detection of Toxoplasma-specific IgG antibody.

[0262] Furthermore, there are several advantages of the recombinantantigen cocktail over the tachyzoite antigen for use in detection of IgGantibodies. First, the antigens are purified, and the amount of eachantigen loaded into the immunoassay can be accurately determined andstandardized, e.g., protein concentration. This minimizes between lotdifferences commonly observed in kits manufactured with differenttachyzoite antigen lots. Hence, different lots of kits manufactured withdifferent antigen cocktail lots will be very consistent from lot to lot.Secondly, mouse or human monoclonal antibodies to the individualrecombinant Toxo antigens are used to monitor coating of the proteins tothe solid phase. This further ensures that each lot produced isconsistent. Third, the true clinical sensitivity of the assay using thepurified antigens will be higher by virtue of the fact of the higherspecific activity of the purified antigens. Finally, kits manufacturedwith the antigen cocktail are more stable during storage over time, andthe performance of the assay using these antigens remains consistentover the shelf life of the assay. Kits manufactured with the tachyzoiteantigen are not as stable and their performance may vary over time.

[0263] Additionally, there are many advantages of using a cocktail overusing a single antigen alone. For example, an immune response toinfection varies by individual. Some individuals produce antibodies toP35 and not to P30 early in infection (acute toxoplasmosis), whereassome individuals produce antibodies to P30 and not to P35 later ininfection (chronic toxoplasmosis). Thus, the antigen cocktail of thepresent invention will detect both groups of individuals.

[0264] Moreover, immune responses vary with time. For example, oneindividual may produce antibodies against P35 first and then laterproduce antibodies to only P30. Thus, the present cocktail will detectboth types of “positive” individuals.

[0265] Furthermore, individuals may be infected with different Toxoserotypes, strains or isolates. Thus, the immune response may be suchthat multiple antigens are needed to detect the presence of allantibodies being produced. Again, the present cocktail allows for suchdetection.

[0266] Also, it is known from previous Western Blot experiments withtachyzoite proteins that the immune response to Toxoplasma is directedagainst several antigens. Once again, the present antigen cocktail willallow for the detection of all antibodies produced in response to theseantigens.

[0267] Step E: Evaluation of the AxSYM® Toxo IgM v2 Assays

[0268] All specimens in Groups 1-3 were tested by the AxSYM® Toxo IgM v2assays (P30del3C, P30del4C, and P30MIX1) and by the AxSYM® Toxo IgMassay. A receiver operator characteristic (ROC) was used to assist thedetermination of the preliminary cutoff for the AxSYM® Toxo IgM v2assays (Index value ≧0.6) (Zweig, H M (1993) Clin. Chem. 39:561-577). Inaddition, an equivocal zone of Index value 0.500-0.599 was introduced toaccount for assay imprecision. The performance of the recombinantantigen based AxSYM® Toxo IgM v2 assays was compared to the tachyzoiteantigen based AxSYM® Toxo IgM assay and is shown in Tables 8-10. TABLE 8Evaluation of the AxSYM ® Toxo IgM v2 P30del13X assay

[0269] TABLE 9 Evaluation of the AxSYM ® Toxo IgM v2 P30del14C assay

[0270] TABLE 10 Evaluation of the AxSYM ® Toxo IgM v2 P30MIX1 assay

[0271] As can be seen from Tables 8-10, the AxSYM® Toxo IgM v2 assayusing the genetically engineered antigen P30 antigen (P30del3C,P30del4C, or P30MIX1) is both a sensitive and specific assay for thedetection of Toxoplasma-specific IgG as demonstrated by the overall highrelative diagnostic sensitivity (range=97.2%-100%), specificity (range94.5%-95.7%), and agreement (range=95.4%-96.4%). The geneticallyengineered Toxo recombinant P30 antigen (P30del3C, P30del4C, orP30MIX1), in combination with the AxSYM® Toxo IgM v2 assay, is bothnecessary and sufficient to replace the tachyzoite for the detection ofToxoplasma-specific IgM antibody.

[0272] Furthermore, there are several advantages of the geneticallyengineered recombinant Toxo P30 antigen over the tachyzoite antigen foruse in detection of IgM antibodies. First, the antigen is purified, andthe amount of antigen loaded into the immunoassay can be accuratelydetermined and standardized, e.g., protein concentration. This minimizeslot-to-to differences commonly observed in kits manufactured withdifferent tachyzoite antigen lots. Hence, different lots of kitsmanufactured with different recombinant antigen lots will be veryconsistent from lot to lot. Secondly, mouse or human monoclonalantibodies to the recombinant Toxo antigen are used to monitor coatingof the proteins to the solid phase. This further ensures that each lotproduced is consistent. Third, the true clinical sensitivity of theassay using the purified antigens will be higher by virtue of the factof the higher specific activity of the purified antigen. Finally, kitsmanufactured with the recombinant antigen are more stable during storageover time, and the performance of the assay using this antigen remainsconsistent over the shelf life of the assay. Kits manufactured with thetachyzoite antigen are not as stable and their performance may vary overtime.

1. An isolated nucleotide sequence or fragment thereof comprising orcomplementary to a nucleotide sequence having at least 70% nucleotidesequence identity to a nucleotide sequence selected from the groupconsisting of SEQ ID NO:22, SEQ ID NO:27 and SEQ ID NO:63:
 2. Anisolated nucleotide sequence or fragment thereof encoding a polypeptide,wherein said polypeptide has at least 70% amino acid sequence identityto an amino acid sequence selected from the group consisting of SEQ IDNO:23, SEQ ID NO:28 and SEQ ID NO:64.
 3. A purified polypeptide encodedby said isolated nucleotide sequence of claim 1 or
 2. 4. A purifiedpolypeptide or fragment thereof having at least 70% amino acid sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64.
 5. A purified polypeptideor fragment thereof comprising an amino acid sequence having 1-6 aminoacids added to the C-terminus of the amino acid sequence of SEQ IDNO:28.
 6. A purified polypeptide or fragment thereof comprising an aminoacid sequence in which at least one of the five C-terminal cysteineamino acids of the amino acid sequence of SEQ ID NO:23 is substitutedwith alanine.
 7. A polyclonal or monoclonal antibody directed againstsaid purified polypeptide of claim 4, 5 or
 6. 8. A compositioncomprising a polypeptide, wherein said polypeptide comprises an aminoacid sequence selected from the group consisting of SEQ ID NO:23, SEQ IDNO:28 and SEQ ID NO:64.
 9. The composition of claim 8 further comprisingP35 of Toxoplasma gondii.
 10. The composition of claim 8 or 9 whereinsaid composition is a diagnostic reagent.
 11. The composition of claim 8wherein said polypeptide is produced by recombinant or synthetic means.12. A method for detecting the presence of IgM antibodies to Toxoplasmagondii in a test sample comprising the steps of: a) contacting said testsample suspected of containing said IgM antibodies with a compositioncomprising a polypeptide, wherein said polypeptide comprises an aminoacid sequence having at least 70% amino acid identity to an amino acidsequence selected from the group consisting of SEQ ID NO:23, SEQ IDNO:28 and SEQ ID NO:64; and b) detecting the presence of polypeptide/IgMantibody complexes, wherein presence of said complexes indicatespresence of said IgM antibodies in said test sample.
 13. A method fordetecting the presence of IgM antibodies to Toxoplasma gondii in a testsample comprising the steps of: a) contacting said test sample suspectedof containing said IgM antibodies with a composition comprising apolypeptide, wherein said polypeptide comprises an amino acid sequencehaving at least 70% amino acid identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:23, SEQ ID NO:28 and SEQID NO:64, for a time and under conditions sufficient for the formationof IgM antibody/antigen complexes; b) adding a conjugate to theresulting IgM antibody/antigen complexes for a time and under conditionssufficient to allow said conjugate to bind to the bound antibody,wherein said conjugate comprises an antibody attached to asignal-generating compound capable of generating a detectable signal;and c) detecting presence of IgM antibodies which may be present in saidtest sample by detecting presence of a signal generated by saidsignal-generating compound.
 14. A method for detecting the presence ofIgG antibodies to Toxoplasma gondii in a test sample comprising thesteps of: a) contacting said test sample suspected of containing saidIgG antibodies with a composition comprising: 1) a polypeptide, whereinsaid polypeptide comprises an amino acid sequence having at least 70%amino acid identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64 and 2) P35;and b) detecting presence of antigen/IgG antibody complexes, presence ofsaid complexes indicating presence of said IgG antibodies in said testsample.
 15. A method for detecting the presence of IgG antibodies toToxoplasma gondii in a test sample comprising the steps of: a)contacting said test sample suspected of containing said IgG antibodieswith a composition comprising: 1) a polypeptide, wherein saidpolypeptide comprises an amino acid sequence having at least 70% aminoacid identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64 and 2) P35,for a time and under conditions sufficient for formation of IgGantibody/antigen complexes; b) adding a conjugate to resulting IgGantibody/antigen complexes for a time and under conditions sufficient toallow said conjugate to bind to bound antibody, wherein said conjugatecomprises an antibody attached to a signal-generating compound capableof generating a detectable signal; and c) detecting IgG antibodies whichmay be present in said test sample by detecting presence of a signalgenerated by said signal-generating compound.
 16. A method for detectingthe presence of IgM antibodies to Toxoplasma gondii in a test samplecomprising the steps of: a) contacting said test sample suspected ofcontaining said IgM antibodies with anti-antibody specific for said IgMantibodies for a time and under conditions sufficient to allow forformation of anti-antibody/IgM antibody complexes; b) adding a conjugateto resulting anti-antibody/IgM antibody complexes for a time and underconditions sufficient to allow said conjugate to bind to bound antibody,wherein said conjugate comprises a polypeptide, wherein said polypeptidecomprises an amino acid sequence having at least 70% amino acid sequenceidentity to an amino acid sequence selected from the group consisting ofSEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64, attached to a signalgenerating compound capable of generating a detectable signal; and c)detecting IgM antibodies which may be present in said test sample bydetecting presence of a signal generated by said signal-generatingcompound.
 17. A method for detecting the presence of IgG antibodies toToxoplasma gondii in a test sample comprising the steps of: a)contacting said test sample suspected of containing said IgG antibodieswith anti-antibody specific for said IgG antibodies for a time and underconditions sufficient to allow for formation of anti-antibody/IgGantibody complexes; b) adding a conjugate to resulting anti-antibody/IgGantibody complexes for a time and under conditions sufficient to allowsaid conjugate to bind to bound antibody, wherein said conjugatecomprises: 1) a polypeptide, wherein said polypeptide comprises an aminoacid sequence having at least 70% amino acid sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NO:23,SEQ ID NO:28 and SEQ ID NO:64, and 2) P35, each attached to asignal-generating compound capable of generating a detectable signal;and c) detecting IgG antibodies which may be present in said test sampleby detecting presence of a signal generated by each of saidsignal-generating compounds.
 18. A vaccine comprising: a) at least onepolypeptide selected from the group consisting of: 1) a polypeptide,wherein said polypeptide comprises amino acid sequence having at least70% amino acid sequence identity to an amino acid sequence selected fromthe group consisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64 and2) P35, and b) a pharmaceutically acceptable adjuvant.
 19. A kit fordetermining the presence of IgM antibodies to Toxoplasma gondii in atest sample comprising: a) a composition comprising a polypeptide,wherein said polypeptide comprises an amino acid sequence having atleast 70% amino acid sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NO:23, SEQ ID NO:28 and SEQID NO:64; and b) a conjugate comprising an antibody attached to asignal-generating compound capable of generating a detectable signal.20. A kit for determining the presence of IgG antibodies to Toxoplasmagondii in a test sample comprising: a) a composition comprising: 1) apolypeptide, wherein said polypeptide comprises an amino acid sequencehaving at least 70% amino acid sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO:23, SEQ IDNO:28 and SEQ ID NO:64 and 2) P35; and b) a conjugate comprising anantibody attached to a signal-generating compound capable of generatinga detectable signal.
 21. A kit for determining the presence of IgMantibodies to Toxoplasma gondii in a test sample comprising: a) ananti-antibody specific for IgM antibody; and b) a composition comprisinga polypeptide, wherein said polypeptide comprises an amino acid sequencehaving at least 70% amino acid sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO:23, SEQ IDNO:28 and SEQ ID NO:64.
 22. A kit for determining the presence of IgMantibodies to Toxoplasma gondii in a test sample comprising: a) ananti-antibody specific for IgM antibody; b) a conjugate comprising: 1) acomposition comprising a polypeptide, wherein said polypeptide comprisesan amino acid sequence having at least 70% amino acid sequence identityto an amino acid sequence selected from the group consisting of SEQ IDNO:23, SEQ ID NO:28 and SEQ ID NO:64, attached to 2) a signal-generatingcompound capable of generating a detectable signal.
 23. A kit fordetermining the presence of IgG antibodies to Toxoplasma gondii in atest sample comprising: a) an anti-antibody specific for IgG antibody;and b) a composition comprising: 1) a polypeptide, wherein saidpolypeptide comprises an amino acid sequence having at least 70% aminoacid sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64 and 2) P35.24. A kit for determining the presence of IgG antibodies to Toxoplasmagondii in a test sample comprising: a) an anti-antibody specific for IgGantibody; b) a conjugate comprising: 1) a polypeptide, wherein saidpolypeptide comprises an amino acid sequence having at least 70% aminoacid sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64, and 2) P35,each attached to a signal generating compound capable of generating adetectable signal.
 25. A method for detecting the presence of IgMantibodies to Toxoplasma gondii in a test sample comprising the stepsof: (a) contacting said test sample suspected of containing IgMantibodies with anti-antibody specific for said IgM antibodies for atime and under conditions sufficient to allow for formation ofanti-antibody IgM complexes; (b) adding antigen to resultinganti-antibody/IgM complexes for a time and under conditions sufficientto allow said antigen to bind to bound IgM antibody, said antigencomprising a polypeptide, wherein said polypeptide comprises an aminoacid sequence having at least 70% amino acid sequence identity to anamino acid sequence selected from the group consisting of SEQ ID NO:23,SEQ ID NO:28 and SEQ ID NO:64; and (c) adding a conjugate to resultinganti-antibody/IgM/antigen complexes, said conjugate comprising acomposition comprising monoclonal or polyclonal antibody attached to asignal-generating compound capable of generating a detectable signal;and (d) detecting IgM antibodies which may be present in said testsample by detecting a signal generated by said signal-generatingcompound.
 26. A method for detecting the presence of IgG antibodies toToxoplasma gondii in a test sample comprising the steps of: (a)contacting said test sample suspected of containing IgG antibodies withanti-antibody specific for said IgG antibodies for a time and underconditions sufficient to allow for formation of anti-antibody IgGcomplexes; (b) adding antigen to resulting anti-antibody/IgG complexesfor a time and under conditions sufficient to allow said antigen to bindto bound IgG antibody, said antigen comprising a mixture of 1) apolypeptide, wherein said polypeptide comprises an amino acid sequencehaving at least 70% amino acid sequence identity to an amino acidsequence selected from the group consisting of SEQ ID NO:23, SEQ IDNO:28 and SEQ ID NO:64, and 2) P35; (c) adding a conjugate to resultinganti-antibody/IgG/antigen complexes, said conjugate comprising acomposition comprising monoclonal or polyclonal antibody attached to asignal-generating compound capable of generating a detectable signal;and (d) detecting IgG antibodies which may be present in said testsample by detecting a signal generated by said signal-generatingcompound.
 27. A method for detecting the presence of IgM and IgGantibodies to Toxoplasma gondii in a test sample comprising the stepsof: a) contacting said test sample suspected of containing said IgM andIgG antibodies with a composition comprising 1) a polypeptide, whereinsaid polypeptide comprises an amino acid sequence having at least 70%amino acid sequence identity to an amino acid sequence selected from thegroup consisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64, and 2)P35, for a time and under conditions sufficient for the formation of IgMantibody/antigen complexes and IgG antibody/antigen complexes; b) addinga conjugate to the resulting IgM antibody/antigen complexes and IgGantibody/antigen complexes for a time and under conditions sufficient toallow said conjugate to bind to the bound IgM and IgG antibody, whereinsaid conjugate comprises an antibody attached to a signal-generatingcompound capable of generating a detectable signal; and c) detecting thepresence of IgM and IgG antibodies which may be present in said testsample by detecting a signal generated by said signal-generatingcompound.
 28. A method for detecting the presence of IgM and IgGantibodies to Toxoplasma gondii in a test sample comprising the stepsof: a) contacting said test sample suspected of containing said IgM andIgG antibodies with anti-antibody specific for said IgM antibodies andsaid IgG antibodies for a time and under conditions sufficient to allowfor formation of anti-antibody/IgM antibody complexes andanti-antibody/IgG antibody complexes; b) adding a conjugate to resultinganti-antibody/IgM antibody complexes and resulting anti-antibody/IgGantibody complexes for a time and under conditions sufficient to allowsaid conjugate to bind to bound antibody, wherein said conjugatecomprises a composition comprising 1) a polypeptide, wherein saidpolypeptide comprises an amino acid sequence having at least 70% aminoacid sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64, and 2) P35,each attached to a signal-generating compound capable of generating adetectable signal; and c) detecting IgM and IgG antibodies which may bepresent in said test sample by detecting a signal generated by saidsignal-generating compound.
 29. A method for detecting the presence ofIgM and IgG antibodies to Toxoplasma gondii in a test sample comprisingthe steps of: (a) contacting said test sample suspected of containingIgM and IgG antibodies with anti-antibody specific for said IgMantibodies and with anti-antibody specific for said IgG antibodies for atime and under conditions sufficient to allow for formation ofanti-antibody/IgM complexes and anti-antibody/IgG complexes; (b) addingantigen to resulting anti-antibody/IgM complexes and resultinganti-antibody/IgG complexes for a time and under conditions sufficientto allow said antigen to bind to bound IgM antibody, said antigencomprising: 1) a polypeptide selected having an amino acid selected fromthe group consisting of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:64, and2) P35; and (c) adding a conjugate to resultinganti-antibody/IgM/antigen complexes and anti-antibody/IgG/antigencomplexes, said conjugate comprising a composition comprising monoclonalor polyclonal antibody attached to a signal generating compound capableof generating a detectable signal; and (d) detecting IgM and IgGantibodies which may be present in said test sample by detecting asignal generated by said signal generating compound.
 30. A method ofproducing monoclonal antibodies comprising the steps of: a) injecting anon-human mammal with a polypeptide, wherein said polypeptide comprisesan amino acid sequence having at least 70% amino acid sequence identityto an amino acid sequence selected from the group consisting of SEQ IDNO:23, SEQ ID NO:28 and SEQ ID NO:64; b) fusing spleen cells of saidnon-human mammal with myeloma cells in order to generate hybridomas; andc) culturing said hybridomas for a time and under conditions sufficientfor said hybridomas to produce said monoclonal antibodies.
 31. PlasmidpMBP-c2X-ToxoP30del3C(52-300aa).
 32. An isolated nucleotide sequencecomprising or complementary to the nucleotide sequence of SEQ ID NO:20.33. A purified polypeptide comprising the amino acid sequence of SEQ IDNO:21.
 34. Plasmid pMBP-c2X-ToxoP30del4C(52-294aa).
 35. An isolatednucleotide sequence comprising or complementary to the nucleotidesequence of SEQ ID NO:25.
 36. A purified polypeptide comprising theamino acid sequence of SEQ ID NO:26.
 37. Plasmid pMBP-c2X-ToxoP30 MIX1.38. An isolated nucleotide sequence comprising or complementary to thenucleotide sequence of SEQ ID NO:61.
 39. A purified polypeptidecomprising the amino acid sequence of SEQ ID NO:62.